Chimeric cytokine receptors and their use in cell therapy

Abstract

Immune effector cells are provided that express a chimeric cytokine receptor comprising a first extracellular antigen-binding domain, a first transmembrane domain, and a cytokine receptor intracellular domain, and a functional exogenous receptor comprising a second extracellular antigen-binding domain, a second transmembrane domain, and an intracellular signaling domain.

Description

[Technical Field]

[0001] cross reference This application claims priority to International Patent Application No. PCT / CN2021 / 088111, filed April 19, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing entitled "P11132-PCT.220419.Sequence Listing.txt", created on April 15, 2022, having a size of 10,10,034 bytes, which was submitted herewith in text form.

[0003] 1. Field The present disclosure relates to chimeric cytokine receptors, engineered immune effector cells comprising same, and methods of use thereof. The present disclosure further relates to the activation and expansion of cells for therapeutic applications, and in particular to T cell immunotherapy based on functional exogenous receptors (e.g., chimeric antigen receptors). [Background technology]

[0004] 2.Background Adoptive transfer of T cells represents an innovative treatment strategy for cancer. However, various obstacles limit the effectiveness of CAR-T cell therapy and / or prevent its widespread application, especially in solid tumors. The FDA has approved four CAR-T products, all of which are autologous CAR-T cells, derived from a patient's own T cells. Allogeneic CAR-T cells derived from healthy donors offer a more commercially viable off-the-shelf option and have the potential to treat a wider range of patients. Although allogeneic CAR-T cells have many advantages over autologous CAR-T cells, the duration of responses to the former is shorter than that of the latter. Therefore, there is a need in the art for next-generation allogeneic CAR-T cells with improved persistence. Summary of the Invention

[0005] 3. Overview In one aspect, the description provides an immune effector cell that expresses (i) a chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain, and (ii) optionally, a functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain.

[0006] In one aspect, the description provides an immune effector cell that expresses (i) a chimeric cytokine receptor comprising (a) a first transmembrane domain, and (b) a cytokine receptor intracellular domain, and (ii) a functional exogenous receptor comprising (a) an extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain.

[0007] In some embodiments, the immune effector cells express (i) a chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain; and (ii) optionally, a functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the immune effector cells express (i) a chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain; and (ii) a functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:29, 135, 141, 143 or 149, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:29, 135, 141, 143 or 149. In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:30-134, 136-140, 142, 144, 146, or 148, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:30-134, 136-140, 142, 144, 146, or 148.In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:150 or 151, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:150 or 151.

[0008] In some embodiments, the immune effector cell further comprises one or more tags. In some embodiments, the tag is linked to the chimeric cytokine receptor and / or the functional exogenous receptor. In some embodiments, the tag linked to the chimeric cytokine receptor is different from the tag linked to the functional exogenous receptor. In some embodiments, the tag linked to the chimeric cytokine receptor and / or the functional exogenous receptor comprises the amino acid sequence of SEQ ID NO:4, 5, or 158-160, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO:4, 5, or 158-160.

[0009] In some embodiments, the extracellular antigen-binding domain binds to an antigen expressed on the surface of a target cell, e.g., a tumor cell. In some embodiments, the extracellular antigen-binding domain is derived from NKG2D or a truncated NKG2D (e.g., the ECD of NKG2D or a truncated NKG2D), or a variant thereof. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6, 156, or 157, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6, 156, or 157. In some embodiments, the extracellular antigen-binding domain is derived from an NKG2D ligand or a variant thereof. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA / B, ULBP-1, ULBP-2, ULBP-5, ULBP-6, and ULBP-3. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more scFvs or one or more sdAbs that bind to an NKG2D ligand. In some embodiments, the extracellular antigen-binding domain is derived from TIGIT or a variant thereof. In some embodiments, the extracellular antigen-binding domain is derived from the ECD of TIGIT. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 145. In some embodiments, the extracellular antigen-binding domain is derived from SIRP-α or a variant thereof. In some embodiments, the extracellular antigen-binding domain is derived from the ECD of SIRP-α. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:147, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:147.In some embodiments, the first or second extracellular antigen-binding domain binds to an antigen expressed on the surface of a target cell, e.g., a tumor cell. In some embodiments, the first or second extracellular antigen-binding domain is derived from NKG2D or a truncated NKG2D (e.g., the ECD of NKG2D or a truncated NKG2D), or a variant thereof. In some embodiments, the first or second extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6, 156, or 157, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6, 156, or 157. In some embodiments, the first or second extracellular antigen-binding domain is derived from an NKG2D ligand or a variant thereof. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA / B, ULBP-1, ULBP-2, ULBP-5, ULBP-6, and ULBP-3. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more scFvs or one or more sdAbs that bind to an NKG2D ligand. In some embodiments, the first or second extracellular antigen-binding domain is derived from TIGIT or a variant thereof. In some embodiments, the first or second extracellular antigen-binding domain is derived from the ECD of TIGIT. In some embodiments, the first or second extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:145, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:145. In some embodiments, the first or second extracellular antigen-binding domain is derived from SIRP-α or a variant thereof, hi some embodiments, the first or second extracellular antigen-binding domain is derived from the ECD of SIRP-α.In some embodiments, the first or second extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:147, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:147.

[0010] In some embodiments, the first transmembrane domain comprises a Janus kinase (JAK) binding domain. In some embodiments, the JAK binding domain is derived from the group consisting of EPOR, GHR, TPOR, and variants thereof. In some embodiments, the JAK binding domain is derived from TPOR, e.g., comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:7. In some embodiments, the JAK binding domain is derived from EPOR. In some embodiments, the JAK binding domain is derived from GHR.

[0011] In some embodiments, the cytokine receptor intracellular domain is derived from one or more cytokine receptors selected from the group consisting of IL2Rα, IL7Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, and combinations thereof. In some embodiments, the cytokine receptor intracellular domain is IL-7Rα, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL-18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL23R-1, IL23R-2, IL23R-3, GM-CSFRα, GM-CSFRβ -1, GM-CSFRβ-2, IL9R-1, IL9R-2, IL9R-3, IL-7Rα-IL12Rβ1, IL-7Rα-IL15Rα, IL-7Rα-IL15Rβ-1, IL-7 Rα-IL15Rβ-2, IL7Rα-IL21R-1, IL7Rα-IL21R-2, IL7Rα-IL21R-3, IL7Rα-IL23R-2, IL7Rα-GMCSFRα, IL- 7Rα-GM-CSFRβ-1, IL-7Rα-GM-CSFRβ-2, IL12Rβ1-IL15Rα, IL12Rβ1-IL15Rβ-1, IL12Rβ1-IL15Rβ-2, IL 12Rβ1-IL-21R-1, IL12Rβ1-IL-21R-2, IL12Rβ1-IL-21R-3, IL12Rβ1-IL-23R-2, IL12Rβ1-GM-CSFRα, IL 12Rβ1-GM-CSFRβ-1, IL12Rβ1-GM-CSFRβ-2, IL12Rβ2-IL15Rα, IL12Rβ2-IL15Rβ-1, IL12Rβ2-IL15Rβ-2, IL12Rβ2-IL-21R-1, IL12Rβ2-IL-21R-2, IL12Rβ2-IL-21R-3, IL12Rβ2-IL-23R-2, IL12Rβ2-GM-CSFRα,IL12Rβ2-GM-CSFRβ-1 IL12Rβ2-GM-CSFRβ-2 IL15Rα-IL-21R-1 IL-21R-2, IL15Rα-IL-21R-3, IL15Rα-IL-23R-2, IL15Rα-GM-CSFRα, IL15R α-GM-CSFRβ-1, IL15Rα-GM-CSFRβ-2, IL15Rβ-1-IL-21R-1, L15Rβ-1-IL-2 1R-2, IL15Rβ-1-IL-21R-3, IL15Rβ-1-IL-23R-2, IL15Rβ-1-GM-CSFRα, IL1 5Rβ-1-GM-CSFRβ-1, IL15Rβ-1-GM-CSFRβ-2, IL15Rβ-2-IL-21R-1, IL15Rβ -2-IL-21R-2, IL15Rβ-2-IL-21R-3, L15Rβ-2-IL-23R-2, IL15Rβ-2-GM-CSF Rα, IL15Rβ-2-GM-CSFRβ-1, IL15Rβ-2-GM-CSFRβ-2, IL-21R-1-IL-23R-2, IL-21R-1-GM-CSFRα, IL-21R-1-GM-CSFRβ-1, IL-21R-1-GM-CSFRβ-2, IL-2 1R-2-IL-23R-2, IL-21R-2-GM-CSFRα, IL-21R-2-GM-CSFRβ-1, IL-21R-2-GM-CSFRβ-2, IL-21R-3-IL-23R-2, IL-21R-3-GM-CSFRα, IL-21R-3-GM-CSF Rβ-1, IL-21R-3-GM-CSFRβ-2, IL-23R-2-GM-CSFRα, IL-23R-2-GM-CSFRβ- 1, IL-23R-2-GM-CSFRβ-2, IL-7Rα-IL-9R-2, IL12Rβ1-IL-9R-2, IL12Rβ2-I L-9R-2, IL15Rα-IL-9R-2, IL15Rβ-1-IL-9R-2, IL15Rβ-2-IL-9R-2, IL-2 R-1-IL-9R-2, IL-21R-2-IL-9R-2, IL-21R-3-IL-9R-2, IL-23R-2-IL-9R-2 GM-CSFRα-IL-9R-2, GM-CSFRβ-1-IL-9R-2, GM-CSFRβ-2-IL-9R-2, IL-7R α-IL-12Rβ2 is a strong mechanism of action You can find the rest of the rest of the range.The cytokine receptor intracellular domain comprises an amino acid sequence selected from SEQ ID NOs: 8-28, or a region having an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NOs: 8-28. In specific embodiments, the cytokine receptor intracellular domain comprises one or more amino acid sequences of SEQ ID NOs: 8, 10, 14-17, or 20-28.

[0012] In some embodiments, the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen coupler (TAC)-like chimeric receptor. In some embodiments, the functional exogenous receptor is a CAR. In other embodiments, the functional exogenous receptor is a TCR. In some embodiments, the functional exogenous receptor is a CAR, as described in more detail in Section 5.2.2. For example, the extracellular domain of the CAR may be in any form, including, for example, a single CAR, a dual CAR, a tandem CAR, or a split CAR. The extracellular domain of the functional exogenous receptor can bind to an antigen expressed on a target cell (e.g., a tumor cell). In some embodiments, the CAR binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of GPC3, AFP, claudin 18.2, CD19, CD20, CD22, BCMA, GD2, DLL3, MSLN, CD30, CLL1, and CD33. In some specific embodiments, a CAR in accordance with the present disclosure binds to GPC3. In some even more specific embodiments, a CAR comprises the amino acid sequence of SEQ ID NO:29 or 135. In some specific embodiments, a CAR in accordance with the present disclosure binds to CD19. In some even more specific embodiments, a CAR comprises the amino acid sequence of SEQ ID NO:141. In some specific embodiments, a CAR in accordance with the present disclosure binds to CD33. In some even more specific embodiments, a CAR comprises the amino acid sequence of SEQ ID NO:143.

[0013] In some embodiments, the second transmembrane domain is derived from a molecule selected from the group consisting of CD8, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the second transmembrane domain is from CD8α or CD28.

[0014] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is from CD3ζ.

[0015] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain.

[0016] In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

[0017] In some embodiments, the functional exogenous receptor further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is from CD8α.

[0018] In some embodiments, the chimeric cytokine receptor and / or functional exogenous receptor further comprises a signal peptide located at the N-terminus of the chimeric cytokine receptor and / or functional exogenous receptor. In some embodiments, the signal peptide is from CD8α.

[0019] In some embodiments, the immune effector cell is a T cell, a natural killer (NK) cell, a NK T cell, a macrophage cell, a peripheral blood mononuclear cell (PBMC), a monocyte, a neutrophil, or an eosinophil. In some embodiments, the T cell is a cytotoxic T cell, a helper T cell, a natural killer T cell, an αβ T cell, or a γδ T cell.

[0020] In another aspect, the description provides a polypeptide comprising: (i) a chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain; and (ii) optionally, a functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain. In one aspect, the description provides a polypeptide comprising: (i) a chimeric cytokine receptor comprising (a) a first transmembrane domain, and (b) a cytokine receptor intracellular domain; and (ii) a functional exogenous receptor comprising (a) an extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain.

[0021] In some embodiments, the polypeptide comprises: (i) a chimeric cytokine receptor comprising: (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof; (b) a first transmembrane domain; and (c) a cytokine receptor intracellular domain; and (ii) optionally, a functional exogenous receptor comprising: (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the polypeptide comprises: (i) a chimeric cytokine receptor comprising: (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof; (b) a first transmembrane domain; and (c) a cytokine receptor intracellular domain; and (ii) a functional exogenous receptor comprising: (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:29, 135, 141, 143 or 149, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:29, 135, 141, 143 or 149. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 30-134, 136-140, 142, 144, 146, or 148, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 30-134, 136-140, 142, 144, 146, or 148. In some embodiments, the polypeptide further comprises one or more tags.In some embodiments, a tag is linked to the chimeric cytokine receptor and / or the functional exogenous receptor. In some embodiments, the tag linked to the chimeric cytokine receptor is different from the tag linked to the functional exogenous receptor. In some embodiments, the tag linked to the chimeric cytokine receptor comprises the amino acid sequence of SEQ ID NO:4, 5, or 158-160, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:4, 5, or 158-160. In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:150 or 151, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:150 or 151.

[0022] In some embodiments, the first extracellular antigen-binding domain binds to an antigen expressed on the surface of a target cell, e.g., a tumor cell. In some embodiments, the first extracellular antigen-binding domain is derived from NKG2D or a truncated NKG2D (e.g., the ECD of NKG2D or a truncated NKG2D), or a variant thereof. In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6, 156, or 157, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6, 156, or 157. In some embodiments, the first extracellular antigen-binding domain is derived from a truncated NKG2D (amino acid positions 89-216, SEQ ID NO:157). In some embodiments, the first extracellular antigen-binding domain is derived from truncated NKG2D (amino acid positions 98-216, SEQ ID NO:156). In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the first extracellular antigen-binding domain is derived from TIGIT (e.g., the ECD of TIGIT). In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:145. In some embodiments, the first extracellular antigen-binding domain is derived from SIRP-α (e.g., the ECD of SIRP-α). In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:147. In some embodiments, the first extracellular antigen-binding domain is derived from an antibody or antigen-binding fragment thereof that targets an NKG2D ligand or a variant thereof. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA / B, ULBP-1, ULBP-2, ULBP-5, ULBP-6, and ULBP-3. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more scFvs or one or more sdAbs that bind to an NKG2D ligand.

[0023] In some embodiments, the first transmembrane domain comprises a Janus kinase (JAK) binding domain. In some embodiments, the JAK binding domain is derived from the group consisting of EPOR, GHR, TPOR, or a variant thereof. In some embodiments, the JAK binding domain is derived from TPOR, e.g., comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:7. In some embodiments, the JAK binding domain is derived from EPOR. In some embodiments, the JAK binding domain is derived from GHR.

[0024] In some embodiments, the cytokine receptor intracellular domain is derived from one or more cytokine receptors selected from the group consisting of IL2Rα, IL7Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, and combinations thereof. In some embodiments, the cytokine receptor intracellular domain is IL-7Rα, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL-18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL23R-1, IL23R-2, IL23R-3, GM-CSFRα, GM-CSFRβ- 1, GM-CSFRβ-2, IL9R-1, IL9R-2, IL9R-3, IL-7Rα-IL12Rβ1, IL-7Rα-IL15Rα, IL-7Rα-IL15Rβ-1, IL-7Rα -IL15Rβ-2, IL-7Rα-IL21R-1, IL-7Rα-IL21R-2, IL-7Rα-IL21R-3, IL-7Rα-IL23R-2, IL-7Rα-GMCSFRα, I L-7Rα-GM-CSFRβ-1, IL-7Rα-GM-CSFRβ-2, IL12Rβ1-IL15Rα, IL12Rβ1-IL15Rβ-1, IL12Rβ1-IL15Rβ-2, I L12Rβ1-IL-21R-1, IL12Rβ1-IL-21R-2, IL12Rβ1-IL-21R-3, IL12Rβ1-IL-23R-2, IL12Rβ1-GM-CSFRα, I L12Rβ1-GM-CSFRβ-1, IL12Rβ1-GM-CSFRβ-2, IL12Rβ2-IL15Rα, IL12Rβ2-IL15Rβ-1, IL12Rβ2-IL15Rβ-2 , IL12Rβ2-IL-21R-1, IL12Rβ2-IL-21R-2, IL12Rβ2-IL-21R-3, IL12Rβ2-IL-23R-2, IL12Rβ2-GM-CSFRα,Derived from a cytokine receptor selected from IL12Rβ2-GM-CSFRβ-1, IL12Rβ2-GM-CSFRβ-2, IL15Rα-IL-21R-1, IL15Rα-IL-21R-2, IL15Rα-IL-21R-3, IL15Rα-IL-23R-2, IL15Rα-GM-CSFRα, IL15Rα-GM-CSFRβ-1, IL15Rα-GM-CSFRβ-2, IL15Rβ-1-IL-21R-1, L15Rβ-1-IL-21R-2, IL15Rβ-1-IL-21R-3, IL15Rβ-1-IL-23R-2, IL15Rβ-1-GM-CSFRα, IL15Rβ-1-GM-CSFRβ-1, IL15Rβ-1-GM-CSFRβ-2, IL15Rβ-2-IL-21R-1, IL15Rβ-2-IL-21R-2, IL15Rβ-2-IL-21R-3, L15Rβ-2-IL-23R-2, IL15Rβ-2-GM-CSFRα, IL15Rβ-2-GM-CSFRβ-1, IL15Rβ-2-GM-CSFRβ-2, IL-21R-1-IL-23R-2, IL-21R-1-GM-CSFRα, IL-21R-1-GM-CSFRβ-1, IL-21R-1-GM-CSFRβ-2, IL-21R-2-IL-23R-2, IL-21R-2-GM-CSFRα, IL-21R-2-GM-CSFRβ-1, IL-21R-2-GM-CSFRβ-2, IL-21R-3-IL-23R-2, IL-21R-3-GM-CSFRα, IL-21R-3-GM-CSFRβ-1, IL-21R-3-GM-CSFRβ-2, IL-23R-2-GM-CSFR α, IL-23R-2-GM-CSFRβ-1, IL-23R-2-GM-CSFRβ-2, IL-7Rα-IL-9R-2, IL12Rβ1-IL-9R-2, IL12Rβ2-IL-9R-2, IL15Rα-IL-9R-2, IL15Rβ-1-IL-9R-2, IL15Rβ-2-IL-9R-2, IL-21R-1-IL-9R-2, IL-21R-2-IL-9R-2, IL-21R-3-IL-9R-2, IL-23R-2-IL-9R-2, GM-CSFRα-IL-9R-2, GM-CSFRβ-1-IL-9R-2, GM-CSFRβ-2-IL-9R-2, IL-7Rα-IL-12Rβ2, or variants thereof. In some embodiments, the cytokine receptor intracellular domain is,The cytokine receptor intracellular domain comprises an amino acid sequence selected from SEQ ID NOs: 8-28, or a region having an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NOs: 8-28. In specific embodiments, the cytokine receptor intracellular domain comprises one or more amino acid sequences of SEQ ID NOs: 8, 10, 14-17, or 20-28.

[0025] In some embodiments, the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen conjugate (TAC)-like chimeric receptor. In some embodiments, the functional exogenous receptor is a CAR. In other embodiments, the functional exogenous receptor is a TCR. In some embodiments, the CAR is a single CAR, a dual CAR, a tandem CAR, or a split CAR. In some embodiments, the CAR is as described in Section 5.2.2. In some specific embodiments, a CAR in accordance with the present disclosure binds to a tumor-associated antigen. In some specific embodiments, a CAR in accordance with the present disclosure binds to GPC3, CD19, or CD33. In some more specific embodiments, a CAR according to the present disclosure comprises the amino acid sequence of SEQ ID NO: 29, 135, 141, 143, or 149, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 29, 135, 141, 143, or 149.

[0026] In some embodiments, the second transmembrane domain is derived from a molecule selected from the group consisting of CD8, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the second transmembrane domain is from CD8α or CD28.

[0027] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is from CD3ζ.

[0028] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

[0029] In some embodiments, the functional exogenous receptor further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is from CD8α.

[0030] In some embodiments, the chimeric cytokine receptor and / or functional exogenous receptor further comprises a signal peptide located at the N-terminus of the chimeric cytokine receptor and / or functional exogenous receptor. In some embodiments, the signal peptide is from CD8α.

[0031] In some embodiments, the chimeric cytokine receptor and the functional exogenous receptor are linked to each other via a peptide linker, hi some embodiments, the peptide linker is a 2A self-cleaving peptide selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof.

[0032] In some embodiments, the polypeptide further comprises one or more tags. In some embodiments, the tag is linked to the chimeric cytokine receptor and / or the functional exogenous receptor. In some embodiments, the tag linked to the chimeric cytokine receptor is different from the tag linked to the functional exogenous receptor. In some embodiments, the tag linked to the chimeric cytokine receptor and / or the functional exogenous receptor comprises the amino acid sequence of SEQ ID NO:4, 5, or 158-160, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO:4, 5, or 158-160.

[0033] In another aspect, the present specification provides an isolated nucleic acid, the isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide according to the present specification. In yet another aspect, the present specification provides an isolated nucleic acid, the isolated nucleic acid comprising: (i) a first region encoding a chimeric cytokine receptor, the chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain; and (ii) optionally, a second region encoding a functional exogenous receptor, the functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain. In yet another aspect, the present specification provides a vector comprising the isolated nucleic acid according to the present specification.

[0034] In yet another aspect, the description provides a method for producing an immune effector cell, the method comprising introducing a nucleic acid or vector according to the description into an immune cell. In yet another aspect, the description provides a method for producing an immune effector cell, the method comprising introducing into an immune cell a composition comprising: (i) a first nucleic acid encoding a chimeric cytokine receptor, the chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain, and (ii) optionally, a second nucleic acid encoding a functional exogenous receptor, the functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain.

[0035] In yet another aspect, the present description provides immune effector cells produced according to the methods herein.

[0036] In yet another aspect, the present description provides a pharmaceutical composition comprising an immune effector cell, polypeptide, nucleic acid, or vector according to the present description and a pharmaceutically acceptable carrier.

[0037] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to the present disclosure.

[0038] In yet another aspect, the present disclosure provides a disease or disorder, including cancer, inflammatory or autoimmune diseases. In some embodiments, the cancer is a solid cancer or a blood cancer. In some embodiments, the cancer is liver cancer, lymphoma, acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

[0039] In another aspect, the description provides a chimeric cytokine receptor comprising (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) a cytokine receptor intracellular domain. In some embodiments, the extracellular antigen-binding domain is derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT, SIRP-α, or a variant thereof. In some embodiments, the extracellular antigen-binding domain is derived from the extracellular domain of NKG2D or a variant thereof, and optionally, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6, 156, or 157, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6, 156, or 157. In other embodiments, the extracellular domain comprises an antibody or antigen-binding fragment thereof that binds to an NKG2D ligand. In some embodiments, the extracellular domain comprises one or more scFvs or one or more sdAbs that bind to an NKG2D ligand. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA / B, ULBP-1, ULBP-2, 5, 6, and ULBP-3. In some embodiments, the extracellular antigen-binding domain is derived from the extracellular domain of an immune checkpoint. In some embodiments, the immune checkpoint is selected from the group consisting of PD-1, CTLA4, or variants thereof. In some embodiments, the immune checkpoint is PD-1. In some embodiments, the immune checkpoint is CTLA4.

[0040] In another aspect, the description provides an immune effector cell, wherein the immune effector cell expresses a chimeric cytokine receptor according to the description. [Brief explanation of the drawings]

[0041] 4. Brief description of the drawings [Figure 1A]1A-1B show exemplary constructs of CARs or TCRs armored with NKG2D, truncated NKG2D, or mutated NKG2D chimeric cytokine receptors. Figure 1A is a schematic diagram of a CAR armored with NKG2D, truncated NKG2D, or mutated NKG2D chimeric cytokine receptors. [Figure 1B] 1A-1B show exemplary constructs of CARs or TCRs armored with NKG2D, truncated NKG2D, or mutated NKG2D chimeric cytokine receptors. Figure 1B is a schematic diagram of a TCR armored with NKG2D, truncated NKG2D, or mutated NKG2D chimeric cytokine receptors. [Figure 2A] 2A-2B show exemplary constructs of a CAR or TCR armed with a chimeric cytokine receptor comprising a binding domain that targets an NKG2D ligand. Figure 2A is a schematic showing a CAR armed with a chimeric cytokine receptor comprising a binding domain that targets an NKG2D ligand. [Figure 2B] 2A-2B are diagrams depicting exemplary constructs of a CAR or TCR armed with a chimeric cytokine receptor comprising a binding domain that targets an NKG2D ligand. Figure 2B is a schematic diagram showing a TCR armed with a chimeric cytokine receptor comprising a binding domain that targets an NKG2D ligand. [Figure 3] Figures 3A-3B show exemplary constructs of CARs or TCRs armed with different cytokine receptors. Figure 3A shows a CAR armed with NKG2D or a mutated NKG2D chimeric cytokine receptor. Figure 3B shows a TCR armed with NKG2D or a mutated NKG2D chimeric cytokine receptor. [Figure 4] FIG. 1 shows surface expression of NKG2D ligands on the HCC cell line huh7. [Figure 5]FIG. 1 shows the long-term cytotoxicity of anti-GPC3 CAR cells or anti-GPC3 CAR γδ T cells armed with two cytokine receptors of an NKG2D chimera co-cultured with huh7 cells. [Figure 6] This figure shows T cell proliferation in long-term co-culture of huh7 cells with anti-GPC3 CAR T cells or anti-GPC3 CAR γδ T cells armed with two cytokine receptors of an NKG2D chimera. [Figure 7] FIG. 1 shows the long-term cytotoxicity of NKG2D chimeric single cytokine receptor-armed anti-GPC3 CAR γδ T cells co-cultured with huh7 cells. [Figure 8] Figure 1 shows T cell proliferation in long-term co-culture of NKG2D chimeric single cytokine receptor-armed anti-GPC3 CAR γδ T cells with huh7 cells. [Figure 9] FIG. 1 shows the long-term cytotoxicity of anti-GPC3 CAR γδ T cells armed with NKG2D chimeric dual cytokine receptors co-cultured with huh7 cells. [Figure 10] Figure 1 shows T cell proliferation in long-term co-culture of anti-GPC3 CAR γδ T cells armed with two cytokine receptors of NKG2D chimera with huh7 cells. [Figure 11] FIG. 1 shows the long-term cytotoxicity of anti-GPC3 CAR γδ T cells armed with NKG2D chimeric dual cytokine receptors co-cultured with huh7 cells. [Figure 12] Figure 1 shows T cell proliferation in long-term co-culture of anti-GPC3 CAR γδ T cells armed with two cytokine receptors of NKG2D chimera with huh7 cells. [Figure 13] Figure 1 shows the long-term cytotoxicity of anti-GPC3 CAR αβ T cells armed with NKG2D chimeric dual cytokine receptors co-cultured with huh7 cells. [Figure 14]Figure 1 shows T cell proliferation in long-term co-culture of anti-GPC3 CAR αβ T cells armed with two cytokine receptors of NKG2D chimera with huh7 cells. [Figure 15] Figure 1 shows the long-term cytotoxicity of anti-GPC3 CAR αβ T cells armed with NKG2D chimeric dual cytokine receptors co-cultured with huh7 cells. [Figure 16] Figure 1 shows T cell proliferation in long-term co-culture of anti-GPC3 CAR αβ T cells armed with two cytokine receptors of NKG2D chimera with huh7 cells. [Figure 17] Figure 1 shows long-term cytotoxicity of truncated NKG2D chimeric dual cytokine receptor armed anti-GPC3 CAR αβ T cells co-cultured with huh7 cells. [Figure 18] Figure 1 shows T cell proliferation in long-term co-culture of anti-GPC3 CAR αβ T cells armed with two cytokine receptors of a truncated NKG2D chimera with huh7 cells. [Figure 19] Figure 1 shows the long-term cytotoxicity of anti-GPC3 CAR γδ T cells armed with two cytokine receptors of anti-MIC-A / B scFv chimera co-cultured with huh7 cells. [Figure 20] Figure 1 shows T cell proliferation in long-term co-culture of anti-GPC3 CAR γδ T cells armed with two cytokine receptors of anti-MIC-A / B scFv chimera with huh7 cells. [Figure 21] FIG. 10 shows the anti-tumor effect of anti-GPC3 CAR γδ T cells or anti-GPC3 CAR γδ T cells armed with two cytokine receptors of an NKG2D chimera in a huh7 xenograft model. [Figure 22] FIG. 10 shows long-term cytotoxicity of NKG2D chimeric dual cytokine receptor armed Construct 185 CAR αβ T cells co-cultured with huh7 cells. [Figure 23]Figure 1 shows T cell proliferation in long-term co-culture of NKG2D chimeric dual cytokine receptor armed Construct 185 CAR αβ T cells with huh7 cells. [Figure 24] FIG. 10 shows long-term cytotoxicity of NKG2D chimeric dual cytokine receptor armed CTL-019 CAR γδ T cells co-cultured with Raji cells. [Figure 25] Figure 1 shows T cell proliferation in long-term co-culture of NKG2D chimeric dual cytokine receptor armed CTL-019 CAR γδ T cells co-cultured with Raji cells. [Figure 26] Figure 1 shows the long-term cytotoxicity of NKG2D chimeric dual cytokine receptor armed CTL-019 CAR αβ T cells co-cultured with Raji cells. [Figure 27] Figure 1 shows T cell proliferation in long-term co-culture of NKG2D chimeric dual cytokine receptor armed CTL-019 CAR αβ T cells co-cultured with Raji cells. [Figure 28] Figure showing long-term cytotoxicity of AS67190 CAR αβ T cells armed with NKG2D chimeric dual cytokine receptors co-cultured with U937 cells. [Figure 29] Figure showing T cell proliferation in long-term co-culture of NKG2D chimeric dual cytokine receptor armed AS67190 CAR αβ T cells co-cultured with U937 cells. [Figure 30] Figure 1 shows the long-term cytotoxicity of anti-GPC3 CAR γδ T cells armed with TIGIT chimeric dual cytokine receptors co-cultured with huh7 cells. [Figure 31] Figure 1 shows T cell proliferation in long-term co-culture of TIGIT chimeric dual cytokine receptor-armed anti-GPC3 CAR γδ T cells co-cultured with huh7 cells. [Figure 32]This figure shows the anti-tumor effect of anti-GPC3 CAR γδ T cells or anti-GPC3 CAR γδ T cells armed with two cytokine receptors of a TIGIT chimera in a huh7 xenograft model. [Figure 33] Figure showing long-term cytotoxicity of AS67190 CAR γδ T cells armed with two cytokine receptors of a SIRP-α chimera co-cultured with U937 cells. [Figure 34] Figure 1 shows T cell proliferation in long-term co-culture of AS67190 CAR γδ T cells armed with two cytokine receptors of a SIRP-α chimera co-cultured with U937 cells. [Figure 35] Figure 1 shows T cell proliferation of NKG2D CAR or BM CAR αβT cells in culture. [Figure 36] FIG. 1 shows T cell viability of NKG2D or BM CAR αβ T cells in culture. [Figure 37] FIG. 10 shows long-term cytotoxicity of BM CAR αβ T cells armed with NKG2D deleted or constitutively active chimeric dual cytokine receptors co-cultured with huh7 cells. [Figure 38] Figure 1 shows T cell proliferation in long-term co-cultures of BM CAR αβT cells armed with NKG2D deleted or constitutively active chimeric dual cytokine receptors co-cultured with huh7 cells. DETAILED DESCRIPTION OF THE INVENTION

[0042] 5. Detailed Description The present disclosure is based in part on the surprising discovery of improved function and properties of engineered immune cells that express chimeric cytokine receptors.

[0043] Engineered CAR-T cells have been engineered to secrete cytokines to promote their survival and / or greater activity. However, the effects of constitutive or inducible cytokine secretion are systemic and not specific to CAR-T cells. They also induce the expansion of endogenous immune cells such as NK cells, NKT cells, dendritic cells (DCs), macrophages, and endogenous CD8 T cells (see Avanzi MP et al., Cell Rep. 15; 23 (7): 2130-2141 (2015)). Because excessive activation of the immune system can be fatal, a more ideal and safer engineering approach is needed.

[0044] The present disclosure provides compositions and methods for genetically modifying immune cells to express chimeric cytokine receptors that respond to ligands specifically expressed in tumor cells. Upon binding of the chimeric cytokine receptor to the ligand, downstream intracellular cytokine signals are activated and uniquely enhance the functional activity of the genetically modified immune cells, which avoids widespread activation of the immune system. Engineered cells according to the present disclosure exhibit enhanced proliferation and efficacy in treating, for example, cancer and infectious diseases.

[0045] 5.1. Definition The techniques and procedures described or referenced herein include those generally well understood by those skilled in the art and / or commonly employed using conventional methods, such as those widely used methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (eds. Ausubel et al., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (eds. An, 2009), Monoclonal Antibodies: Methods and Protocols (eds. Albitar, 2010), and Antibody Engineering, Vol. 1 and Vol. 2 (eds. Kontermann and Dubel, 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For the purposes of this specification, the following terminology applies, and where appropriate, terms used in the singular also include the plural, and vice versa. In the event that any explanation of a term provided herein contradicts any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0046] The terms "antibody," "immunoglobulin," or "Ig" may be used interchangeably herein and are used in the broadest sense, and specifically refer to, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, single-domain antibodies (e.g., V HH) and fragments thereof (e.g., domain antibodies). Antibodies can be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species (e.g., mouse, rabbit, llama, etc.). The term "antibody" is intended to include polypeptide products of B cells in the immunoglobulin family, which have the ability to bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (ed. by Borrebaeck, 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those derived from Camelidae species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. These functional fragments refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which they are derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies according to the present specification include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody).Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2nd ed., 1990). Antibodies according to the present invention can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. Antibodies can be agonist or antagonist antibodies. Antibodies can be neither agonist nor antagonist.

[0047] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In some embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0048] An "intact" antibody is one that comprises an antigen-binding site and a CL and at least heavy chain constant regions CH1, CH2, and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In some embodiments, an intact antibody has one or more effector functions.

[0049] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0050] As used herein, a "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain and is capable of binding to an antigen. Single domain antibodies are those comprising the V domains described herein. H The single domain antibody comprises an H domain. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains, such as antibodies from Camelidae species (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. For example, as described herein, single domain antibodies may be derived from antibodies produced from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other species outside of Camelidae can produce heavy chain antibodies that naturally lack light chains, and V domains derived from such other species may be used. H H is within the scope of the present disclosure. In some embodiments, a single domain antibody (e.g., V HH) has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0051] The terms "binds" or "binding," "specifically binds," or "specific for" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target, which determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is about 10% less than the binding of the antibody to the target as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding may include, but is not required to include, exclusive binding. In another embodiment, the term "binds" or "binding" refers to interactions between molecules, including, for example, forming a complex.

[0052] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which a binding molecule (e.g., an antibody containing a single-chain antibody sequence) can specifically bind. An epitope may be a linear epitope or a conformational, non-linear or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope may be a continuous amino acid of the polypeptide ("linear" epitope), or an epitope may contain amino acids from two or more non-contiguous regions of the polypeptide ("conformational", "non-linear" or "discontinuous" epitope). One of ordinary skill in the art will generally understand that a linear epitope may or may not depend on secondary, tertiary or quaternary structure. For example, in some embodiments, the binding molecule binds to a group of amino acids regardless of whether they are folded into their native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues constituting the epitope assume a specific conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0053] "Percent amino acid sequence identity (%)" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in a particular peptide or polypeptide sequence when sequence alignment is performed to achieve the maximum percent sequence identity and gaps are introduced if necessary, and no conservative substitutions are considered part of the sequence identity. Alignment can be achieved and the percent amino acid sequence identity determined using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (商標) (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared.

[0054] As used herein, the term "chimeric cytokine receptor" or CCR refers to a molecule comprising a cytokine receptor endodomain, a transmembrane domain, and a heterologous ligand-binding exodomain. The heterologous exodomain binds to a ligand other than the cytokine for which the cytokine receptor from which the endodomain is derived is selective. Thus, the ligand specificity of a cytokine receptor can be altered by grafting a heterologous binding specificity.

[0055] In some embodiments, the CCR comprising a transmembrane domain comprises a Janus kinase (JAK) binding domain. In some embodiments, the JAK binding domain is derived from the group consisting of EPOR, GHR, TPOR, or variants thereof. In some embodiments, the JAK binding domain comprises the amino acid sequence of SEQ ID NO:7.

[0056] In some embodiments, the CCR comprises an intracellular domain derived from a cytokine receptor or an intracellular domain of a composition, e.g., IL2Rα, IL7Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. The chimeric cytokine receptor may further comprise an extracellular domain and a transmembrane domain.

[0057] In some embodiments, the CCR comprises a tag, wherein the tag comprises an amino acid sequence of SEQ ID NO:4, 5 or 158-160, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO:4, 5 or 158-160.

[0058] As used herein, the term "functional exogenous receptor" refers to an exogenous receptor (e.g., a recombinant or engineered TCR, such as a cTCR, a TAC-like chimeric receptor, or a CAR) that retains its biological activity after introduction into an immune effector cell (e.g., a T cell). Biological activity includes, but is not limited to, the ability of the exogenous receptor to specifically bind to a molecule, appropriately transduce downstream signals, e.g., induce cell proliferation, cytokine production, and / or execute regulatory or cytolytic effector functions.

[0059] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transfer one or more antigens into immune effector cells, such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." Chimeric molecules contain one or more antigen-binding moieties (e.g., single-domain antibodies or scFvs) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). Typically, a CAR is composed of an antigen-binding moiety, a transmembrane domain, and an intracellular domain. The intracellular domain typically contains a signaling chain with an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some cases, the intracellular domain further contains the intracellular portion of at least one additional costimulatory domain (e.g., CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or hematopoietic cell signaling factor (DAP10)). In the context of this application, the terms "cytoplasmic domain," "intracellular domain," and "intracellular signaling domain" may be used interchangeably. In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. In some embodiments, a CAR is a single CAR. In some embodiments, a CAR is a dual CAR. In some embodiments, a CAR is a tandem CAR. In some embodiments, a CAR is a split CAR. "CAR-T cell" refers to a T cell expressing a CAR. As used herein, the term "single CAR" includes chimeric molecules that comprise a single antigen-binding moiety (e.g., a single-domain antibody or scFv) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). Typically, a single CAR may comprise a monospecific antigen-binding moiety, a transmembrane domain, and an intracellular domain.

[0060] As used herein, the term "tandem CAR" includes chimeric molecules that contain more than one antigen-binding moiety (e.g., 2, 4, or 6 single domain antibodies or scFvs) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). Typically, a tandem CAR contains a monospecific, bivalent antigen-binding moiety (e.g., two identical Vs that bind to GPC3). H H domain) or a multispecific (e.g., bispecific) bivalent antigen-binding moiety (e.g., two different VH domains that bind to GPC3). H H domain or one V that binds to GPC3 H H domain and another V that binds to other molecules other than GPC3 H In some embodiments, the tandem CAR of the present disclosure may comprise an extracellular antigen-binding domain, which further comprises a second binding domain that binds to a second antigen, and which is different from GPC3.

[0061] As used herein, the term "dual CAR" includes two separate CARs (which may be bispecific), two tandem CARs (each CAR may be monospecific or bispecific), or one single CAR and one tandem CAR (which may be monospecific or bispecific). A dual CAR may have a first CAR and a second CAR, each of which has a costimulatory domain (e.g., CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and DAP10) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). A dual CAR may be a combination of any two anti-GPC3 CARs, where each of the first CAR and the second CAR may be a single CAR or a tandem CAR, i.e., single CAR / single CAR, single CAR / tandem CAR, or tandem CAR / tandem CAR. The level of dual CAR T cell signaling can be adjusted by manipulating the intracellular domains of each of the first and second CARs. For example, the intracellular domain of each of the first CAR and the second CAR may comprise a costimulatory domain (e.g., CD28, CD137 (4-1BB), ICOS, OX40 (CD134), CD27, and / or DAP10), and / or a signaling domain from a T cell receptor (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). For example, a dual CAR of the present disclosure may comprise a first CAR and a second CAR, each of which comprises a costimulatory domain and an intracellular domain of a signaling domain from a T cell receptor. Thus, when the dual CAR binds to an antigen (e.g., bispecifically), a T cell signal can be transmitted through two signaling domains from the T cell receptor.

[0062] As used herein, the term "split CAR" differs from a dual CAR in that in a split CAR, a first CAR may comprise a costimulatory domain but lack a signaling domain from a T cell receptor, whereas a second CAR may comprise a signaling domain from a T cell receptor but lack a costimulatory domain. In some embodiments, a split CAR system of the present disclosure may comprise a first intracellular signaling domain comprising a primary intracellular signaling domain of an immune effector cell and a second intracellular signaling domain comprising a costimulatory signaling domain.

[0063] As used herein, the term "recombinant or engineered TCR" refers to a peptide contained as a functional exogenous receptor according to the present invention and expressed in immune cells. The function of a recombinant or engineered TCR may include, for example, redirecting the immune activity of an immune cell against a desired type of cell, such as cancer cells and infected cells bearing a specific marker on their surface. They can replace or co-express with an endogenous TCR. In some embodiments, such recombinant TCRs are single-chain TCRs containing an open reading frame in which the variable Vα and Vβ domains are paired using a protein linker. This involves molecular cloning of TCR genes known to be specific for a selected antigen, and then introducing these chains into T cells, typically via a retroviral vector. Expression of the cloned TCRα and TCRβ genes then confers functional specificity to the transduced T cell, determined by the pairing of these new genes. Components of a recombinant or engineered TCR can be any functional subunit of a TCR, such as recombinant TCRα and TCRβ, encoded by an exogenous polynucleotide sequence introduced into a cell.

[0064] In some embodiments, the functional exogenous receptor according to the present invention is a chimeric TCR (cTCR), which simultaneously possesses antigen-binding and T cell activation functions. For example, the cTCR may comprise: (a) an extracellular ligand-binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3, CD19, or CLL1); (b) an optional linker; (c) an optional extracellular domain or portion thereof of a first TCR subunit (e.g., CD3ε); (d) a transmembrane domain comprising the transmembrane domain of a second TCR subunit (e.g., CD3ε); and (e) an intracellular signaling domain comprising the intracellular signaling domain of a third TCR subunit (e.g., CD3ε), wherein the first, second, and third TCR subunits are all selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ. In some embodiments, the first, second, and third TCR subunits are the same (e.g., all are CD3ε). In some embodiments, the first, second, and third TCR subunits are different. In some embodiments, the cTCR further comprises a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the cTCR further comprises a signal peptide located at the N-terminus of the cTCR, for example, a signal peptide derived from CD8α.

[0065] In some embodiments, the functional exogenous receptor is a T cell antigen conjugate (TAC), e.g., comprising: (a) an extracellular ligand binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3, CD19, or CLL1); (b) an optional first linker; (c) an extracellular TCR binding domain that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3ε); (d) an optional second linker; (e) an optional extracellular domain or portion thereof of a first TCR co-receptor (e.g., CD4); (f) a transmembrane domain comprising the transmembrane domain of a second TCR co-receptor (e.g., CD4); and (g) an optional intracellular signaling domain comprising the intracellular signaling domain of a third TCR co-receptor (e.g., CD4). wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ, and wherein the first, second, and third TCR co-receptors are all selected from the group consisting of CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are the same. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC further comprises a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the TAC further comprises a signal peptide located at the N-terminus of the TAC, for example, a signal peptide derived from CD8α. In some embodiments, the extracellular ligand-binding domain is located at the N-terminus of the extracellular TCR binding domain. In some embodiments, the extracellular ligand-binding domain is located at the C-terminus of the extracellular TCR binding domain.

[0066] In some embodiments, the functional exogenous receptor is a TAC-like chimeric receptor, for example, comprising: (a) an extracellular ligand-binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3); (b) an optional first linker; (c) an extracellular TCR-binding domain that specifically recognizes the extracellular domain of a first TCR subunit (e.g., TCRα); (d) an optional second linker; and (e) a second TCR subunit (e.g., TCRα). (f) a transmembrane domain comprising the transmembrane domain of a third TCR subunit (e.g., CD3ε); and (g) an optional intracellular signaling domain comprising the intracellular signaling domain of a fourth TCR subunit (e.g., CD3ε), wherein the first, second, third, and fourth TCR subunits are all selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ. In some embodiments, the second, third, and fourth TCR subunits are the same. In some embodiments, the first, second, third, and fourth TCR subunits are different. In some embodiments, the second, third, and fourth TCR subunits are the same but different from the first TCR subunit. In some embodiments, the extracellular ligand-binding domain is located N-terminal to the extracellular TCR binding domain. In some embodiments, the extracellular ligand-binding domain is located at the C-terminus of the extracellular TCR-binding domain. In some embodiments, the TAC-like chimeric receptor further comprises a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the TAC-like chimeric receptor further comprises a signal peptide, for example, a signal peptide derived from CD8α, located at the N-terminus of the TAC-like chimeric receptor.

[0067] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. A polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms further encompass amino acid polymers that have been modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition further includes, for example, polypeptides containing one or more amino acid analogs (including, but not limited to, unnatural amino acids) and other modifications known in the art. It should be understood that, because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, a "polypeptide" can exist as a single chain or two or more associated chains.

[0068] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or a synthetic reaction. A polynucleotide may also include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, generally single-stranded, synthetic polynucleotide, usually (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure may include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced.

[0069] An "isolated nucleic acid" is a nucleic acid (e.g., RNA, DNA, or mixture of nucleic acids) that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Note that an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding the single-chain antibodies or antibodies described herein are isolated or purified. The term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule may include an isolated form of the molecule. In particular, an "isolated" nucleic acid molecule encoding a CAR or a CAR-armed CCR, as described herein, is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced.

[0070] The term "vector" refers to a substance for carrying or containing a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, for introducing the nucleic acid sequence into a host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain a selection sequence or a marker capable of stable integration into a host cell chromosome. A vector may also contain one or more selection marker genes and appropriate expression control sequences. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as known in the art. When two or more nucleic acid molecules are to be coexpressed (e.g., both antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acids may be operably linked to a common expression control sequence or to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of a nucleic acid molecule into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblots for expression of gene products, or other suitable analytical methods to test for expression of the introduced nucleic acid sequence or its corresponding gene product. It should be understood by those skilled in the art that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0071] As used herein, the term "host" refers to an animal, for example, a mammal (e.g., a human).

[0072] As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the host cell genome.

[0073] As used herein, the term "autologous" refers to any material derived from the same individual, where the material is later reintroduced into that individual.

[0074] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0075] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0076] As used herein, the term "immune response" includes the response of a cell of the immune system (e.g., a B cell, a T cell, or a monocyte) to a stimulus. In some embodiments, the response has specificity for a particular antigen (an "antigen-specific response"). In some embodiments, the immune response is a T cell response, e.g., a CD4 + Response or CD8 + In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0077] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeia for use in animals, more specifically, humans. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of a pharmaceutical formulation and suitable for contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic reaction, immunogenicity, or other problems or complications, consistent with a reasonable benefit-risk ratio. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005, Handbook of Pharmaceutical Excipients, 6th Edition; Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; Gibson, ed., CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed, hi some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0078] "Carrier" or "excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0079] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an engineered immune effector cell, or a therapeutic molecule, or pharmaceutical composition comprising an agent and engineered immune effector cell, provided herein, sufficient to bring about a desired result.

[0080] The terms "subject" and "patient" may be used interchangeably. As used herein, in some embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0081] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body into the body of a patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art.

[0082] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition through the administration of one or more therapies. Treatment may be determined by assessing whether one or more symptoms associated with the underlying condition are reduced, ameliorated, and / or alleviated, such that the patient is observed to improve, even though the patient may still have the underlying condition. The term "treatment" includes disease control and amelioration.

[0083] The terms "control," "controlling," and "control" refer to the beneficial effect that a subject derives from a therapy that does not necessarily result in a cure of the disease.

[0084] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of a disease, disorder, condition, or associated symptom (e.g., cancer) occurring (or recurring).

[0085] As used herein, "delaying" cancer progression refers to slowing, inhibiting, slowing, extending, stabilizing, and / or slowing the progression of the disease. This delay may have different lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual is free from the disease. A method for "delaying" cancer progression is one that reduces the probability of disease progression within a given time range and / or reduces the extent of disease within a given time range compared to the absence of the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. Cancer progression can be detected using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation studies, arteriography, or biopsy. Progression may also refer to the progression of cancer, which may be initially undetectable, and includes occurrence, recurrence, and seizures.

[0086] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1% or less of a given value or range.

[0087] As used in this disclosure and the claims, the singular forms "a / an" and "the / said" include the plural forms unless the context clearly dictates otherwise.

[0088] It is to be understood that wherever this specification describes an embodiment with the term "comprising," similar embodiments are also provided where described with "consisting of" and / or "consisting essentially of." It is further to be understood that wherever this specification describes an embodiment with the phrase "consisting essentially of," similar embodiments are also provided where described with "consisting of."

[0089] The term "between" used in the phrase "between A and B" or "between A and B" refers to a range that includes A and B.

[0090] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0091] 5.2. Engineered immune effector cells As described in Section 6 below, introducing a chimeric cytokine receptor into immune effector cells expressing one or more functional exogenous receptors can significantly improve the properties / function of the immune effector cells. In some embodiments, the immune effector cells express (i) a chimeric cytokine receptor comprising (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof, (b) a first transmembrane domain, and (c) a cytokine receptor intracellular domain, and (ii) optionally, a functional exogenous receptor comprising (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) an intracellular signaling domain.

[0092] Thus, in one aspect, the description provides a host cell (e.g., an immune effector cell), wherein the host cell comprises a chimeric cytokine receptor comprising: (a) a first extracellular antigen-binding domain; (b) a first transmembrane domain; and (c) a cytokine receptor intracellular domain. In some embodiments, the extracellular domain is derived from the extracellular domain of NKG2D or a variant thereof. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the first extracellular antigen-binding domain is derived from a truncated NKG2D. In some embodiments, the first extracellular antigen-binding domain is derived from a truncated NKG2D (amino acid positions 89-216, SEQ ID NO:157). In some embodiments, the first extracellular antigen-binding domain is derived from a truncated NKG2D (amino acid positions 98-216, SEQ ID NO:156). In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6, 156 or 157.

[0093] In other embodiments, the extracellular domain comprises an antibody or antigen-binding fragment thereof that binds to an NKG2D ligand. In some embodiments, the extracellular domain comprises one or more scFvs that bind to an NKG2D ligand. In other embodiments, the extracellular domain comprises one or more sdAbs (e.g., one or more V H The NKG2D ligand may be selected from the group consisting of, but not limited to, MICA / B, ULBP-1, ULBP-2, 5, 6, and ULBP-3.

[0094] In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:29, 135, 141, 143 or 149, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:29, 135, 141, 143 or 149. In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:30-134, 136-140, 142, 144, 146, or 148, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:30-134, 136-140, 142, 144, 146, or 148. In some embodiments, the immune effector cells comprise the amino acid sequence of SEQ ID NO:150 or 151, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:150 or 151.

[0095] NKG2D is a transmembrane protein that belongs to the NKG2 family of C-type lectin-like receptors and is also known as KLRK1, CD314, D12S2489E, KLR, NKG2-D, NKG2 natural killer group 2D, killer cell lectin-like receptor K1, and killer cell lectin-like receptor K1. NKG2D is encoded by the KLRK1 gene, which is located in the NK gene complex (NKC) on chromosome 6 in mice and chromosome 12 in humans. In humans, it is involved in the expression of NK cells, γδ T cells, and CD8 + It is expressed by αβ T cells. NKG2D recognizes induced self-proteins from the MIC and RAET1 / ULBP families, which are present on the surface of stressed, malignant, and infected cells. NKG2D ligands are induced self-proteins that are completely absent or present at low levels on the surface of normal cells, but are overexpressed by infected, transformed, senescent, and stressed cells. NKG2D sequences are known in the art (see, e.g., OMIM: 611817, homologous gene: 136440, gene card: KLRK1).

[0096] In some embodiments, the first extracellular antigen-binding domain is derived from TIGIT. In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the first extracellular antigen-binding domain is derived from SIRP-α. In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 147.

[0097] In some embodiments, the host cell expresses one or more functional exogenous receptors. The functional exogenous receptor may be, for example, a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric TCR (cTCR), or a chimeric receptor like a T cell antigen conjugate (TAC). In some embodiments, the functional exogenous receptor is a CAR. In some embodiments, the functional exogenous receptor is a TCR. In some embodiments, the functional exogenous receptor is a cTCR. In other embodiments, the functional exogenous receptor is a TAC. Any immune effector cell capable of performing an immune effector function can be used in the present disclosure, including, but not limited to, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0098] In some embodiments, the present specification provides a host cell (e.g., an immune effector cell) comprising any one of the polypeptides, polynucleotides, or vectors described herein (e.g., as provided in the sections below).

[0099] 5.2.1. Chimeric Cytokine Receptors In one aspect, the present disclosure provides a chimeric cytokine receptor, which comprises an intracellular domain derived from one or more cytokine receptors, i.e., a cytokine receptor intracellular domain. The chimeric cytokine receptor according to the present disclosure also comprises an extracellular domain and a transmembrane domain.

[0100] Cytokine receptor intracellular domain In some embodiments, the cytokine receptor utilized in the chimeric cytokine receptor of the present invention is selected from the group consisting of IL2Rα, IL7Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, and combinations thereof.

[0101] In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL2Rα. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL7Rα. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL9R-1. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL9R-2. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL9R-3. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL12Rβ1. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL12Rβ2. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL15Rα. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL15Rβ-1. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL15Rβ-2. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL15Rβ-3. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL-18Rα. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL18Rβ.In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL21R-1. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL21R-2. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL21R-3. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL-10R2. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL22R1. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL23R-1. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL23R-2. In some embodiments, a chimeric cytokine receptor according to the present specification comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL23R-3. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL-27Rα. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from gp130. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL31RA. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from OSMRβ. In some embodiments, a chimeric cytokine receptor according to the present disclosure comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL36R.In some embodiments, a chimeric cytokine receptor provided herein comprises an intracellular domain, wherein the intracellular domain comprises a region derived from IL1RAcP. In some embodiments, a chimeric cytokine receptor provided herein comprises an intracellular domain, wherein the intracellular domain comprises a region derived from GM-CSFRα. In some embodiments, a chimeric cytokine receptor provided herein comprises an intracellular domain, wherein the intracellular domain comprises a region derived from GM-CSFRβ-1. In some embodiments, a chimeric cytokine receptor provided herein comprises an intracellular domain, wherein the intracellular domain comprises a region derived from GM-CSFRβ-2.

[0102] In some embodiments, the intracellular domain of a chimeric cytokine receptor according to the present disclosure comprises two or more (e.g., 2, 3, 4, 5, or more) sequences from IL2Rα, IL7Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.

[0103] For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL2Rα and a region derived from IL7Rα, IL2Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL7Rα and a region derived from IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL9R-1 and a region derived from IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL9R-2 and a region derived from IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL9R-3 and a region derived from IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL12Rβ1 and a region derived from IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL12Rβ2 and a region derived from IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, or variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL15Rα and a region derived from IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL15Rβ-1 and a region derived from IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL15Rβ-2 and a region derived from IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL15Rβ-3 and a region derived from IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL18Rα and a region derived from IL18R, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL18Rβ and a region derived from IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor according to the present disclosure comprises a region derived from IL21R-1 and a region derived from IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL21R-2 and a region derived from IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL21R-3 and a region derived from IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL10R2 and a region derived from IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL22R1 and a region derived from IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL22R1 and a region derived from GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL23R-1 and a region derived from IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL23R-2 and a region derived from IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. The intracellular domain of a chimeric cytokine receptor comprises a region derived from IL23R-3 and a region derived from IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor according to the present disclosure comprises a region derived from IL27Rα and a region derived from gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from gp130 and a region derived from IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL31RA and a region derived from OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from OSMRβ and a region derived from IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, a variant thereof, or a combination thereof. For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL36R and a region derived from IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof.For example, in some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from IL1RAcP and a region derived from GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, GM-CSFRβ-1, GM-CSFRβ-2, variants thereof, or combinations thereof. In some embodiments, the intracellular domain of a chimeric cytokine receptor provided herein comprises a region derived from GM-CSFRβ-1 and a region derived from GM-CSFRβ-2 or a variant thereof.

[0104] Then you have a nice smile on your face Their target molecules are IL-7Rα, IL12Rβ1, and IL12 Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL-18Rα, IL18Rβ, IL21R-1, I L21R-2, IL21R-3, IL23R-1, IL23R-2, IL23R-3, GM-CSFRα, GM-CSFRβ-1, GM- CSFRβ-2, IL9R-1, IL9R-2, IL9R-3, IL-7Rα-IL12Rβ1, IL-7Rα-IL15Rα, IL-7 Rα-IL15Rβ-1, IL-7Rα-IL15Rβ-2, IL-7Rα-IL21R-1, IL-7Rα-IL21R-2, IL-7 Rα-IL21R-3, IL-7Rα-IL23R-2, IL-7Rα-GMCSFRα, IL-7Rα-GM-CSFRβ-1, IL- 7Rα-GM-CSFRβ-2, IL12Rβ1-IL15Rα; 2, IL12Rβ1-IL-21R-1, IL12Rβ1-IL-21R-2, IL12Rβ1-IL-21R-3, IL12Rβ1-IL -23R-2, IL12Rβ1-GM-CSFRα, IL12Rβ1-GM-CSFRβ-1, IL12Rβ1-GM-CSFRβ-2. IL12Rβ2-IL15Rα, IL12Rβ2-IL15Rβ-1, IL12Rβ2-IL15Rβ-2, IL12Rβ2-IL-21 R-1, IL12Rβ2-IL-21R-2, IL12Rβ2-IL-21R-3, IL12Rβ2-IL-23R-2, IL12Rβ2 -GM-CSFRα IL12Rβ2-GM-CSFRβ-1 IL12Rβ2-GM-CSFRβ-2 IL15Rα-IL-21R- 1, IL15Rα-IL-21R-2, IL15Rα-IL-21R-3, IL15Rα-IL-23R-2, IL15Rα-GM-CS FRα, IL15Rα-GM-CSFRβ-1, IL15Rα-GM-CSFRβ-2, IL15Rβ-1-IL-21R-1, L15R β-1-IL-21R-2, IL15Rβ-1-IL-21R-3, IL15Rβ-1-IL-23R-2, IL15Rβ-1-GM-C SFRα, IL15Rβ-1-GM-CSFRβ-1, IL15Rβ-1-GM-CSFRβ-2, IL15Rβ-2-IL-21R-1Regions derived from IL15Rβ-2-IL-21R-2, IL15Rβ-2-IL-21R-3, L15Rβ-2-IL-23R-2, IL15Rβ-2-GM-CSFRα, IL15Rβ-2-GM-CSFRβ-1, IL15Rβ-2-GM-CSFRβ-2, IL-21R-1-IL-23R-2, IL-21R-1-GM-CSFRα, IL-21R-1-GM-CSFRβ-1, IL-21R-1-GM-CSFRβ-2, IL-21R-2-IL-23R-2, IL-21R-2-GM-CSFRα, IL-21R-2-GM-CSFRβ-1, IL-21R-2-GM-CSFRβ-2, IL-21R-3-IL-23R-2, IL-21R-3-GM-CSFRα, IL-21R-3-GM-CSFRβ-1, IL-21R-3-GM-CSFRβ-2, IL-23R-2-GM-CSFRα, IL-23R-2-GM-CSFRβ-1, IL-23R-2-GM-CSFRβ-2, IL-7Rα-IL-9R-2, IL12Rβ1-IL-9R-2, IL12Rβ2-IL-9R-2, IL15Rα-IL-9R-2, IL15Rβ-1-IL-9R-2, IL15Rβ-2-IL-9R-2, IL-21R-1-IL-9R-2, IL-21R-2-IL-9R-2, IL-21R-3-IL-9R-2, IL-23R-2-IL-9R-2, GM-CSFRα-IL-9R-2, GM-CSFRβ-1-IL-9R-2, GM-CSFRβ-2-IL-9R-2, IL-7Rα-IL-12Rβ2, or variants thereof are included.

[0105] When the intracellular domain of a chimeric cytokine receptor provided herein comprises sequences from two or more cytokine receptors, these sequences may be in any order. In some embodiments, the cytokine receptor intracellular domain comprises a region having an amino acid sequence selected from SEQ ID NOs:8-28, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NOs:8-28. In specific embodiments, the cytokine receptor intracellular domain comprises one or more amino acid sequences of SEQ ID NOs:8, 10, 14-17, or 20-28.

[0106] Transmembrane domain The chimeric cytokine receptors of the present disclosure comprise a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. The transmembrane domain may be derived from natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane (e.g., a eukaryotic cell membrane). A transmembrane domain suitable for the chimeric cytokine receptors described herein can be obtained from a naturally occurring protein. Alternatively, it may be a synthetic, non-natural protein segment, such as a hydrophobin segment, that is thermodynamically stable in a cell membrane.

[0107] Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain may form an α-helix, a complex of α-helices, a β-barrel, or any other stable structure capable of spanning a cellular phospholipid bilayer. Transmembrane domains can also or alternatively be classified based on their topological structure, including the number of times the transmembrane domain spans the membrane and the orientation of the protein. For example, single-pass membrane proteins span the cellular membrane once, while multi-pass membrane proteins span the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their terminal positions relative to the inside and outside of the cell and the topological structure of one or more transmembrane segments. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is located on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is located on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented so that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further classified based on the number of transmembrane segments and the location of the N- and C-termini.

[0108] In some embodiments, the transmembrane domain of the chimeric cytokine receptors described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may be compatible for use in the chimeric cytokine receptors described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helices or beta sheet structures. In some embodiments, the N- and C-termini of a multi-pass membrane protein are on opposite sides of a lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer, while the C-terminus of the protein is on the extracellular side.

[0109] The transmembrane domains used in the chimeric cytokine receptors described herein may further comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, e.g., U.S. Pat. No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0110] A transmembrane domain according to the present specification may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, favors orientation of the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0111] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a chimeric cytokine receptor provided herein comprises a hydrophobic artificial sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminal position of the transmembrane domain. In some embodiments, the transmembrane region comprises primarily hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a polyleucine-alanine sequence. The hydropathy or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydropathy analysis.

[0112] In some embodiments, the transmembrane domain of the chimeric cytokine receptor is selected from the group consisting of the α, β, or ζ chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0113] In some embodiments, the transmembrane domain of the chimeric cytokine receptor of the present invention comprises a Janus kinase (JAK) binding domain. In some embodiments, the JAK binding domain is derived from the group consisting of EPOR, GHR, TPOR, or variants thereof. In some embodiments, the JAK binding domain is derived from TPOR. In some embodiments, the JAK binding domain comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the JAK binding domain is derived from EPOR. In some embodiments, the JAK binding domain is derived from GHR. In some embodiments, in addition to the transmembrane domains described above, the transmembrane domain of the chimeric cytokine receptor of the present invention comprises a JAK binding domain.

[0114] Extracellular domain In some embodiments, the extracellular domain of a chimeric cytokine receptor of the present invention binds to an antigen expressed on the surface of a target cell (eg, a tumor cell or an infected cell).

[0115] The extracellular domain of a chimeric cytokine receptor described herein comprises one or more antigen-binding domains. The extracellular domain of a chimeric cytokine receptor described herein may be in any form, so long as binding of the extracellular domain to its target activates a downstream intracellular cytokine signal, e.g., induces dimerization of the chimeric cytokine receptor. In some embodiments, the extracellular domain is derived from a naturally occurring receptor (e.g., the ECD of the receptor). In other embodiments, the extracellular domain is not derived from a naturally occurring receptor. In some embodiments, the first extracellular antigen-binding domain is derived from NKG2D or a truncated NKG2D (e.g., the ECD of NKG2D or a truncated NKG2D), or a variant thereof. In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6, 156, or 157, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6, 156, or 157. In some embodiments, the first extracellular antigen-binding domain is derived from a truncated NKG2D (amino acid positions 89-216, SEQ ID NO:157). In some embodiments, the first extracellular antigen-binding domain is derived from a truncated NKG2D (amino acid positions 98-216, SEQ ID NO:156). In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the first extracellular antigen-binding domain is derived from TIGIT (e.g., the ECD of TIGIT). In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the first extracellular antigen-binding domain is derived from SIRP-α (e.g., the ECD of SIRP-α). In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 147.In some embodiments, the first extracellular antigen-binding domain is derived from an antibody or antigen-binding fragment thereof that targets an NKG2D ligand or a variant thereof. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA / B, ULBP-1, ULBP-2, 5, 6, and ULBP-3. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more scFvs or one or more sdAbs that bind to an NKG2D ligand. In some embodiments, the extracellular antigen-binding domain is derived from the extracellular domain of an immune checkpoint. In some embodiments, the immune checkpoint is selected from the group consisting of PD-1, CTLA4, or variants thereof. In some embodiments, the immune checkpoint is PD-1. In some embodiments, the immune checkpoint is CTLA4.

[0116] In some embodiments, the extracellular antigen-binding domain of a chimeric cytokine receptor provided herein is monospecific. In other embodiments, the extracellular antigen-binding domain of a chimeric cytokine receptor provided herein is multispecific. In other embodiments, the extracellular antigen-binding domain of a chimeric cytokine receptor provided herein is monovalent. In other embodiments, the extracellular antigen-binding domain of a chimeric cytokine receptor provided herein is multivalent. In some embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains, which are fused to each other by a direct peptide bond or via a peptide linker.

[0117] In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. For example, the binding domain may be derived from a monoclonal antibody (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), an antibody with polyepitopic or monoepitopic specificity, a polyclonal or monovalent antibody, a multivalent antibody, a multispecific antibody formed from at least two intact antibodies (e.g., a bispecific antibody, so long as it exhibits the desired biological activity), a single-chain antibody, a single-domain antibody, and fragments thereof (e.g., domain antibodies). The antibody may be a human antibody, a humanized antibody, a chimeric antibody, and / or an affinity-matured antibody, as well as an antibody from another species (e.g., mouse, rabbit, llama, etc.). In some embodiments, antibodies are intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides, which have the ability to bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (ed. by Borrebaeck, 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). Antibodies further include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those derived from Camelidae species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. These functional fragments refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which they are derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, antibodies according to the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2nd ed., 1990). An antibody according to the present invention can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an agonist antibody or an antagonist antibody. The antibody can be neither an agonist nor an antagonist.

[0118] In a specific embodiment, the extracellular antigen-binding domain of a chimeric cytokine receptor of the invention comprises a single-chain Fv (sFv or scFv). An scFv is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. See Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0119] In another specific embodiment, the extracellular antigen-binding domain of a chimeric cytokine receptor of the invention comprises one or more single domain antibodies (sdAbs). The sdAbs may be of the same or different origin and of the same or different size. An exemplary sdAb comprises a heavy chain variable domain (e.g., V) from an antibody having only a heavy chain. H These include, but are not limited to, single domains derived from conventional four-chain antibodies (e.g., VH or VL), binding molecules naturally devoid of light chains, single domains derived from traditional four-chain antibodies (e.g., VH or VL), humanized antibodies having only heavy chains, human single domain antibodies produced from transgenic mice or rats expressing human heavy chain segments, and single domain scaffolds other than those derived from engineered domains and antibodies. Any sdAb known in the art or developed according to the present disclosure, including the single domain antibodies described above, can be used in constructing the chimeric cytokine receptors described herein. sdAbs may be derived from any species, including, but not limited to, mouse, rat, human, camel, llama, river lamprey, fish, shark, goat, rabbit, and cow. Single domain antibodies contemplated herein further include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0120] In some embodiments, naturally occurring single domain antigen-binding molecules derived from sdAbs are referred to as heavy chain antibodies devoid of light chains (also referred to herein as "antibodies having only heavy chains"). For example, such single domain molecules are disclosed in WO 94 / 04678 and Hamers-Casterman, C. et al. Nature 363:446-448 (1993). For clarity, variable domains derived from heavy chain molecules naturally devoid of light chains are referred to herein as VH regions to distinguish them from conventional VH regions of four-chain immunoglobulins. H This is called H. H H molecules may be derived from antibodies produced in Camelidae species, such as camel, llama, vicuna, dromedary, alpaca, and guanaco. Other non-Camelidae species can produce heavy chain molecules that naturally lack light chains, and such V HH is within the scope of this disclosure. H Humanized versions of H and other modifications and variants are also contemplated and within the scope of this disclosure. In some embodiments, the sdAb is derived from the variable region of an immunoglobulin found in cartilaginous fish. For example, the sdAb may be derived from an immunoglobulin isotype called a novel antigen receptor (NAR), which is found in shark serum. Methods for generating single domain molecules derived from the variable region of an NAR ("IgNAR") are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0121] In some embodiments, a naturally occurring V against a particular antigen or target H The H domain is a member of camelid V H The target antigen or target can be obtained from a (naive or immune) library of H sequences. Such methods may or may not involve screening such a library using the antigen or target, or at least a portion, fragment, antigenic determinant, or epitope thereof, by one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, a (naive or immune) V sequence ... H Improved synthetic or semi-synthetic libraries derived from H libraries, e.g., by techniques described in WO 00 / 43507 such as random mutagenesis and / or CDR shuffling (naive or immune) V H V obtained from the H library H The H library can be used.

[0122] In some embodiments, sdAbs are recombinant, CDR-grafted, humanized, camelized, deimmunized and / or in vitro generated (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by "camelization" of specific amino acid residues in the framework regions. Camelization occurs when one or more amino acid residues in the amino acid sequence from the (naturally occurring) VH domain of a conventional four-chain antibody are replaced by the VH domain of a heavy chain antibody. H This refers to a substitution or replacement of one or more amino acid residues present at one or more corresponding positions in the H domain. This may be performed by methods known in the art and will be apparent to those skilled in the art. Such "camelizing" substitutions are preferably inserted at amino acid positions forming and / or present at the VH-VL interface and / or at so-called Camelidae hallmark residue positions, as defined herein (see, e.g., WO 94 / 04678; Davies and Riechmann FEBS Letters 339: 285-290 (1994); Davies and Riechmann, Protein Engineering 9 (6): 531-537 (1996); Riechmann, J. Mol. Biol. 259: 957-969 (1996); and Riechmann and Muyldermans, J. Immunol. Meth. 231: 25-38 (1999)).

[0123] In some embodiments, the sdAb is a human single domain antibody produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., US20090307787, U.S. Patent No. 8,754,287, US20150289489, US20100122358, and WO 2004049794.

[0124] In some embodiments, single domain antibodies are generated from traditional four-chain antibodies. See, e.g., EP 0 368 684, Ward et al., Nature, 341(6242):544-6 (1989), Holt et al., Trends Biotechnol., 21(11):484-490 (2003), WO 06 / 030220, and WO 06 / 003388.

[0125] In some embodiments, the extracellular antigen-binding domain comprises a humanized antibody or fragment thereof. A humanized antibody may comprise human framework regions and human constant region sequences.

[0126] Humanized antibodies can be produced using a variety of techniques known in the art, including CDR-grafting (European Patent No. EP 239,400, International Publication No. WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, but not limited to, the techniques disclosed in U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 93 / 17105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55(23 Suppl):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent No. US 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced from a non-human source.These non-human amino acid residues are commonly referred to as "import" residues, which are typically taken from an "import" variable domain. For example, humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods described in Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36.

[0127] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. revealed that only about one-third of the residues in the CDRs actually contact the antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In SDR grafting technology, only the SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36:25-34).

[0128] To reduce antigenicity, the selection of human variable domains (both light and heavy chains) used in producing humanized antibodies can be important. For example, the so-called "best-fit" method involves screening the variable domain sequence of a non-human (e.g., rodent) antibody against the entire library of known human variable domain sequences. The human sequence closest to that of a rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308, and Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89, and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequence of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). Alternatively, human germline genes are used as the source of the framework regions.

[0129] In an alternative paradigm based on CDR comparison, called superhumanization, the homology of FR is irrelevant. This method involves comparing non-human sequences with functional human germline gene repertoires. Then, the gene encoding the same or closely related canonical structure as the mouse sequence is selected. Then, among the genes that share the canonical structure with non-human antibodies, the gene with the highest homology within CDR is selected as the FR donor. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0130] It is generally desirable to humanize antibodies to retain their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely effect of the residues on the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues from the recipient and import sequences can be selected and combined to produce the desired antibody characteristic, such as increased affinity for one or more target antigens. Typically, the hypervariable region residues are directly and most significantly involved in influencing antigen binding.

[0131] Another antibody humanization method is based on a metric called Human String Content (HSC), which measures the degree of antibody humanization. This method compares the mouse sequence with the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing the HSC, rather than using a general identity measure, to generate multiple different humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

[0132] In addition to the above methods, empirical methods can also be used to generate and select humanized antibodies. These methods include methods based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries and by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0133] In the FR library method, a set of residue variants is introduced at a specific position within the FR, and the library is screened to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the "vernier" residues identified as likely to contribute to CDR structure (e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99), or a more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[0134] In FR shuffling, entire FRs are combined with non-human CDRs without creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The library can be screened for binding in a two-step process (first VL humanization, then VH). Alternatively, a one-step FR shuffling process can be used. Such methods prove more effective than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0135] The "humaneering" method is based on experimental identification of essential minimum specificity determinants (MSDs), followed by sequential replacement of non-human fragments with a human FR library and evaluation of binding. Starting with the CDR3 regions of the non-human VH and VL chains, other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, are gradually substituted into the human FRs. This method typically results in epitope retention and identification of antibodies from multiple subclasses with different human V-segment CDRs. Humaneering allows the isolation of antibodies with 91% to 96% homology to human germline antibodies (see, e.g., Alfreito, Cambridge Healthtech Institute, 3rd Annual PEGS, The Protein Engineering Summit, 2007).

[0136] "Human engineering" methods involve engineering non-human antibodies or antibody fragments (e.g., murine or chimeric antibodies or antibody fragments) with specific modifications to the antibody's amino acid sequence to create modified antibodies with reduced immunogenicity in humans, while still retaining the required binding characteristics of the original non-human antibody. Typically, the techniques involve classifying amino acid residues in non-human (e.g., murine) antibodies into "low risk," "intermediate risk," or "high risk" residues. Classification is performed using an overall risk / reward calculation that assesses the predicted benefit of a particular substitution (e.g., with respect to human immunogenicity) and the risk that the substitution will affect the folding of the resulting antibody. By aligning amino acid sequences from non-human antibody variable regions with corresponding regions of specific or consensus human antibody sequences, specific human amino acid residues to be substituted at a given position (e.g., low risk or intermediate risk) in the non-human (e.g., murine) antibody sequence can be selected. Amino acid residues at low or intermediate risk positions in the non-human sequence can be substituted for the corresponding residue in the human antibody sequence by alignment. Techniques for producing engineered proteins are described in further detail in Studnicka et al., 1994, Protein Engineering 7:805-14, U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Publication WO 93 / 11794.

[0137] Composite human antibodies include, for example, Composite Human Antibodies (商標) Composite human antibodies can be produced using techniques (Antitope Ltd., Cambridge, United Kingdom). To create composite human antibodies, the variable region sequences are designed from fragments of multiple human antibody variable region sequences to avoid T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibodies. Such antibodies may also contain human constant region sequences, e.g., human light and / or heavy chain constant regions.

[0138] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 2009; 525:405-23, xiv and De Groot et al., Cell. Immunol. 244:148-153 (2006). A deimmunized antibody comprises a variable region from which T cell epitopes have been deleted and a human constant region. Briefly, the VH and VL of an antibody are cloned, followed by identification of T cell epitopes by testing overlapping peptides derived from the antibody VH and VL in a T cell proliferation assay. T cell epitopes are identified using computational methods to identify peptides that bind to human MHC class II. Mutations are introduced into the VH and VL to remove binding to human MHC class II. The mutated VH and VL are then used to generate a deimmunized antibody.

[0139] In certain embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or fragment thereof, for example, the extracellular antigen-binding domain comprises multiple binding domains connected in series (e.g., multiple scFvs). In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or fragment thereof. The term "specificity" refers to the selective recognition of a specific epitope of an antigen by the antigen-binding protein. As used herein, the term "multispecific" indicates that the antigen-binding protein has two or more antigen-binding sites, where at least two bind to different antigens. As used herein, the term "valent" indicates that an antigen-binding protein has a specified number of binding sites. A full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate that the antigen-binding protein has two binding sites, three binding sites, four binding sites, five binding sites, and six binding sites, respectively.

[0140] Multispecific antibodies, such as bispecific antibodies, are antibodies that have binding specificities for at least two different antigens. Methods for producing multispecific antibodies, such as co-expressing two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities, are known in the art (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (ed. by Kontermann, 2011).

[0141] The antibody may be a multivalent antibody (e.g., a tetravalent antibody) having two or more antigen-binding sites, which can be readily produced by recombinant expression of nucleic acids encoding the antibody polypeptide chains. In some embodiments, the multivalent antibody comprises (or is composed of) three to about eight antigen-binding sites. In one such embodiment, the multivalent antibody comprises (or is composed of) four antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein one or more polypeptide chains comprise two or more variable domains. For example, the one or more polypeptide chains may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, one or more polypeptide chains may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain, or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (e.g., four) light chain variable domain polypeptides. For example, the multivalent antibody herein may comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally comprise a CL domain.

[0142] When multiple binding domains are present in the extracellular antigen-binding domain of the chimeric cytokine receptor of the present invention, the various domains can be fused to each other via peptide linkers. In some embodiments, the domains are fused to each other directly without any peptide linkers. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence, depending on the structural and / or functional characteristics of the various domains. Each peptide linker may be independently selected and optimized. The length, flexibility, and / or other properties of one or more peptide linkers used in the chimeric cytokine receptor may have some effect on properties including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. In some embodiments, the peptide linker contains flexible residues (e.g., glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine pair may be a suitable peptide linker.

[0143] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, the sequence of a hinge region from an antibody having only a heavy chain may be used as a linker. See, e.g., WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n (SEQ ID NO:152), (GSGGS)n (SEQ ID NO:153), (GGGS)n (SEQ ID NO:154), and (GGGGS)n (SEQ ID NO:155), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art may be included in the chimeric cytokine receptors herein, for example, as described in WO 2016014789, WO 2015158671, WO 2016102965, US20150299317, WO 2018067992, US 7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference.

[0144] In some embodiments, the extracellular antigen-binding domain provided in the chimeric cytokine receptor of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. Tumors express multiple proteins that can act as target antigens for immune responses, particularly T-cell-mediated immune responses. The antigen targeted by the chimeric cytokine receptor may be an antigen on a single diseased cell or an antigen expressed on different cells that each affect the disease. The antigen targeted by the chimeric cytokine receptor may be directly or indirectly involved in the disease.

[0145] In some embodiments, the antigen of the target cell is an antigen on the surface of a cancer cell, hi some embodiments, the antigen is a tumor-specific antigen, a tumor-associated antigen, or a neoantigen.

[0146] In some embodiments, the target cell is a cancer cell, for example, a cell of the following cancers: adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational choriocarcinoma, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer. In some embodiments, the cancer is adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational choriocarcinoma, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer.

[0147] In some embodiments, the adrenal cancer is adrenocortical carcinoma (ACC), adrenocortical carcinoma, pheochromocytoma, or neuroblastoma. In some embodiments, the anal cancer is squamous cell carcinoma, cloacal carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In some embodiments, the appendix cancer is neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal adenocarcinoma, or signet ring cell adenocarcinoma. In some embodiments, the cholangiocarcinoma is extrahepatic bile duct carcinoma, adenocarcinoma, perihilar cholangiocarcinoma, hilar region cholangiocarcinoma, distal bile duct carcinoma, or intrahepatic cholangiocarcinoma. In some embodiments, the bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, squamous carcinoma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, or sarcoma. In some embodiments, the bone cancer is primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In some embodiments, the brain cancer is astrocytoma, brainstem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary adenocarcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, medulloblastoma, or primary CNS lymphoma. In some embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, breast cancer in situ, breast sarcoma, metaplastic carcinoma, adenoid cystic carcinoma, follicular tumor, angiosarcoma, HER2-positive breast cancer, triple-negative breast cancer, or inflammatory breast cancer. In some embodiments, the cervical cancer is squamous cell carcinoma or adenocarcinoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the gallbladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In some embodiments, the gestational trophoblastic disease (GTD) is hydatidiform mole, gestational trophoblastic neoplasia (GTN), choriocarcinoma, placental site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In some embodiments, the head and neck cancer is laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cancer, oropharyngeal cancer, or tonsillar cancer.In some embodiments, the Hodgkin's lymphoma is classical Hodgkin's lymphoma, nodular sclerosing, mixed cytology, lymphocyte-rich, lymphocyte-reduced, or nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL). In some embodiments, the intestinal cancer is small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments, the kidney cancer is renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvic carcinoma, or renal sarcoma. In some embodiments, the leukemia is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndrome (MDS). In a specific embodiment, the leukemia is AML. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, large cell undifferentiated carcinoma, lung nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, lung carcinoid, mesothelioma, lung sarcomatoid carcinoma, or malignant granular cell lung tumor. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna, amelanotic melanoma, connective tissue proliferative melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments, the multiple myeloma is symptomatic or smoldering myeloma. In some embodiments, the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, a pancreatic neuroendocrine tumor, or a pulmonary neuroendocrine tumor.In some embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of anaplastic large cell lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, follicular lymphoma, cutaneous T-cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small cell lymphocytic lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), precursor T-lymphoblastic leukemia / lymphoma, acute lymphocytic leukemia (ALL), adult T-cell lymphoma / leukemia (ATLL), hairy cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, central nervous system origin lymphoma, and leukemia. Central Nervous System (CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, intranodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, B-cell non-Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, natural killer cell lymphoma, cutaneous T-cell lymphoma, Alibert-Bazin syndrome, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), systemic ALCL, enteropathy-associated T-cell lymphoma (EATL), or hepatosplenic gamma / delta T-cell lymphoma. In some embodiments, the oral cancer is squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granulomatosis, fibroma, granular cell tumor, keratoacanthoma, leiomyoma, osteochondromatosis, lipoma, schwannoma, neurofibroma, papillary cytoma, condyloma acuminata, verrucous xanthomas, pyogenic granuloma, rhabdomyoma, odontogenic tumor, vitiligo, erythema, squamous cell lip cancer, basal cell lip cancer, mouth cancer, gum cancer, or tongue cancer.In some embodiments, the ovarian cancer is epithelial ovarian cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, epithelial ovarian clear cell carcinoma, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumor, primary peritoneal carcinoma, fallopian tube carcinoma, germinoma, teratoma, dysgerminoma, ovarian germ cell carcinoma, endodermal sinus tumor, sex cord-stromal tumor, sex cord-gonadal stromal tumor, ovarian stromal tumor, granular cell tumor, granular-thecal cell tumor, Sertoli-Leydig tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Krukenberg tumor, or ovarian cyst. In some embodiments, the pancreatic cancer is pancreatic exocrine adenocarcinoma, pancreatic endocrine adenocarcinoma, pancreatic adenocarcinoma, insulinoma, or neuroendocrine tumor. In some embodiments, the prostate cancer is prostate adenocarcinoma, prostatic sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor. In some embodiments, the paranasal sinus cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, undifferentiated carcinoma of the nasal and paranasal sinuses, nasal cavity cancer, paranasal sinus cancer, maxillary sinus cancer, ethmoid sinus cancer, or nasopharyngeal carcinoma. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi's sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoechinoderm. In some embodiments, the soft tissue cancer is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid / round cell liposarcoma (MRCL), lipomatous liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, renal sarcoma (RMS), or synovial sarcoma. In some embodiments, the spinal cancer is a metastatic spinal tumor. In some embodiments, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, type I ECL-cell carcinoid, type II ECL-cell carcinoid, or type III ECL-cell carcinoid. In some embodiments, the testicular cancer is seminoma, non-seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratocele, gonadal stromal tumor, testicular Leydig cell tumor, or testicular Sertoli cell tumor.In some embodiments, the pharyngeal cancer is squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, verrucous carcinoma, undifferentiated carcinoma, or lymph node carcinoma. In some embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, Hürthle cell carcinoma, medullary thyroid carcinoma, or undifferentiated carcinoma. In some embodiments, the uterine cancer is endometrial carcinoma, endometrial adenocarcinoma, endometrioid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma. In some embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, the vulvar cancer is squamous cell carcinoma or adenocarcinoma.

[0148] Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T-cell mediated immune response. Exemplary tumor antigens include, but are not limited to, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroid globulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0149] In some embodiments, the cancer antigen is CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EpCAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family antigens, CAGE family antigens, GAGE ​​family antigens, MAGE family antigens, SAGE family antigens, XAGE family antigens, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A, MART-1, Gp100, pmel17, tyrosinase, TRP-1, TRP-2, P polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, or MUC1.

[0150] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of molecules associated with transformation, such as the oncogene HER2 / Neu / ErbB-2. A further group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA).

[0151] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present in other cells in the body. TAA-associated antigens are not unique to tumor cells and can instead be expressed on normal cells under conditions that do not allow for immune tolerance to the antigen to be induced. Expression of an antigen in a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or it can be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0152] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens produced by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0153] Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO- 1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0154] Other non-limiting exemplary targets of chimeric cytokine receptors according to the present disclosure include GPC2, CD276, delta-like protein ligand 3 (DLL3), NY-ESO-1, melanoma-associated antigen 4, survivin, synovial sarcoma X breakpoint 2 protein, CD3, epidermal growth factor receptor (EGFR), erbb2 tyrosine kinase receptor, HER2, CEA, CD66, CD66e, ROR1, ntrkr1 tyrosine kinase receptor, GPC3, mesothelin, glutamate carboxypeptidase II, PMSA, PD-L1, folate receptor alpha, PSCA, mucin 1, HLA antigens (e.g., HLA class I antigen A-2 alpha, HLA class I antigen A-11 alpha, and HLA class II antigens), c-Met, hepatocyte growth factor receptor, K-Ras GTPase (KRAS), IL-15 receptor, Kit tyrosine kinase, PDGF receptor beta, RET tyrosine kinase receptor, Raf 1 protein kinase, Raf B protein kinase, thymidylate synthase, topoisomerase II, Brachyury proteins, Flt3 tyrosine kinase, VEGF, VEGF receptors (VEGF-1 receptor, VEGF-2 receptor, and VEGF-3 receptor), estrogen receptor, neoantigen, human papillomavirus E6, and heat shock proteins.

[0155] In some specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is CD19. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is CD20. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is CD22. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is BCMA. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is VEGFR2. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is FAP. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is EpCam. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is GPC3. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is DLL3. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is CD133. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is IL13Ra. In other specific embodiments, at least one target antigen of the chimeric cytokine receptor of the present invention is EGFRIII. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is EphA2. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is Muc1. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is CD70. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is CD123. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is ROR1. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is PSMA.In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is CD5. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is GD2. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is GAP. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is CD33. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is CEA. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is PSCA. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is Her2. In another specific embodiment, at least one target antigen of the chimeric cytokine receptor of the present invention is mesothelin.

[0156] In some embodiments, the chimeric cytokine receptor provided herein binds to a B cell antigen, such as CD1a, CD1b, CD1c, CD1d, CD2, CD5, CD6, CD9, CD11a, CD11b, CD11c, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD29, CD30, CD31, CD32a, CD32b, CD35, CD37, or CD38. , CD39, CD40, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49b, CD49c, CD49d, CD50, CD52 , CD53, CD54, CD55, CD58, CD60a, CD62L, CD63, CD68, CD69, CD70, CD72, CD73, CD74, CD75, CD75S, CD77, CD 79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85E, CD85I, CD85J, CD86, CD92, CD95, CD97, CD98, CD99, CD 100, CD102, CD108, CD119, CD120a, CD120b, CD121b, CD122, CD124, CD125, CD126, CD130, CD132, CD137, C D138, CD139, CD147, CD148, CD150, CD152, CD162, CD164, CD166, CD167a, CD170, CD171, CD175, CD175s, C D180, CD184, CD185, CD192, CD196, CD197, CD200, CD205, CD201a, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD224, CD225, CD226, CD227, CD229, CD230, CD232, CD252, CD252, CD254, CD255, CD2 56, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD267, CD268, CD269, CD270, CD272, CD274, CD275, CD277, CD279, CD283, CD289, CD290, CD295, CD298, CD300, CD300c, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD314, CD215, CD316, CD317, CD319, CD321, CD327, CD328, CD329, CD338, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD360, CD361, CD362, or CD363 antigen.

[0157] In one embodiment, the target of the chimeric cytokine receptor of the present invention is a pathogen, hi some embodiments, the target cell is a cell that contains a pathogen.

[0158] In some embodiments, the pathogen causes an infectious disease selected from the group consisting of acute flaccid myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, campylobacter infection, carbapenem-resistant infection, chancroid, chikungunya virus infection, chlamydia disease, fish botulism, Clostridium difficile infection, Clostridium perfringens disease, coccidioides fungal infection, coronavirus infection, Covid-19 (SA) RS-CoV-2), Creutzfeldt-Jakob disease / transmissible spongiform encephalopathy, cryptosporidiosis (Crypto), cyclosporiasis, dengue fever types 1, 2, 3, or 4, diphtheria, Shiga toxin-producing Escherichia coli (STEC) infection, Eastern equine encephalitis, hemorrhagic fever (Ebola), ehrlichiosis, encephalitis, arbovirus or parainfectious diseases, non-enterovirus, enterovirus D68 disease (EV-D68), giardiasis, glanders, gonorrhea, granuloma inguinale, Haemophilus influenzae Hepatitis B infection (Hib or H-influenza), hemophagocytic syndrome (HPS), hemolytic uremic syndrome (HUS), hepatitis A (Hep A), hepatitis B (Hep B), hepatitis C (Hep C), hepatitis D (Hep D), hepatitis E (Hep E), herpes zoster (shingles), histoplasmosis infection, human immunodeficiency virus / AIDS (HIV / AIDS), human papillomavirus (HPV), influenza (Flu), Legionnaires' disease, leprosy, leptospirosis, listeriosis (Listeria), Lyme disease, lymphogranuloma venereum (LGV) infection, malaria, measles, melioidosis, meningitis (viral), meningococcal meningitis (meningitis (bacterial)), Middle East respiratory syndrome Group coronavirus disease (MERS-CoV), parotitis, Norwalk virus disease, pediculosis, pelvic inflammatory disease (PID), whooping cough (spastic cough), plague (bubonic plague, septicemic plague, pneumonic plague), pneumococcal infection (pneumonia), acute polio (Polio), Powassan encephalitis, psittacosis, pubic lice infestation, exanthematous pustulosis (smallpox, monkeypox, cowpox), Q fever, rabies, rickettsial infections (Rocky Mountain spotted fever), rubella (Rubella / GermanMeasles), Salmonella gastroenteritis (Salmonella), Scabies, Scombroid food poisoning, Pyemia, Severe Acute Respiratory Syndrome (SARS), Shigella gastroenteritis (Shigella), Smallpox, Methicillin-resistant Staphylococcus aureus (MRSA) infection, Staphylococcal food poisoning Enterotoxin B poisoning (Staphylococcal food poisoning), Vancomycin-intermediate-resistant Staphylococcus aureus infection (VISA), Vancomycin-resistant Staphylococcus aureus infection (VRSA), Group A Streptococcus infection (invasive) (Strep In some embodiments, the infectious disease is acute flaccid myelitis (AFM), group B streptococcal infection (Strep-B), streptococcal toxic shock syndrome (STSS), syphilis (primary, secondary, early latent, late latent, congenital), tetanus infection, trichomoniasis, trichinellosis infection, tuberculosis (TB), latent tuberculosis infection (LTBI), tularemia, group D typhoid, vaginosis, Varicella / Chickenpox, Vibrio cholerae infection (cholera), vibriosis (Vibrio), Ebola hemorrhagic fever, Lasa virus hemorrhagic fever, Marburg hemorrhagic fever, West Nile virus, yellow fever, yersiniosis, and Zika virus infection. In some embodiments, the infectious disease is acute flaccid myelitis (AFM).

[0159] In some embodiments, the pathogen is a bacterium, hi some embodiments, the bacterium is a bacterium of the genus Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydiae, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, or Yersinia.

[0160] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoan, a helminth, or an ectoparasite. In some embodiments, the protozoan is Entamoeba, Giardia, Leishmania, Balantidium, Plasmodium, or Cryptosporidium. In some embodiments, the helminth is a trematode, a tapeworm, an anthelminth, or an ascarid. In some embodiments, the ectoparasite is an arthropod.

[0161] In some embodiments, the pathogen is a virus, hi some embodiments, the virus is a virus of the Adenoviridae, Arenaviridae, Astroviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae families. In some embodiments, the virus is an adenovirus, a coronavirus, a coxsackievirus, an Epstein-Barr virus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus type 2, a cytomegalovirus, a human herpesvirus type 8, a human immunodeficiency virus, an influenza virus, a measles virus, a mumps virus, a human papillomavirus, a parainfluenza virus, a poliovirus, a rabies virus, a respiratory syncytial virus, a rubella virus, or a varicella-zoster virus.

[0162] In some specific embodiments, the extracellular antigen-binding domain of the chimeric cytokine receptor of the invention is derived from NKG2D or a truncated NKG2D (e.g., the ECD of NKG2D or a truncated NKG2D). In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6, 156, or 157, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6, 156, or 157. In other specific embodiments, the extracellular antigen-binding domain of the chimeric cytokine receptor of the invention comprises an antibody or antigen-binding fragment thereof that binds to an NKG2D ligand. In some embodiments, the extracellular domain comprises one or more scFvs that bind to an NKG2D ligand. In other embodiments, the extracellular domain comprises one or more sdAbs (e.g., one or more V H The NKG2D ligand may comprise a cytoplasmic domain (H domain). The NKG2D ligand may be selected from the group consisting of, but not limited to, MICA / B, ULBP-1, ULBP-2, 5, 6, and ULBP-3. In some embodiments, the NKG2D ligand is MICA / B. In some embodiments, the NKG2D ligand is ULBP-1. In some embodiments, the NKG2D ligand is ULBP-2. In some embodiments, the NKG2D ligand is ULBP-5. In some embodiments, the NKG2D ligand is ULBP-6. In some embodiments, the NKG2D ligand is ULBP-3.

[0163] The percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be determined using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100 and wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches may be performed, for example, with the XBLAST program parameters set to score 50 and word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST, described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997), can be used. Alternatively, PSI BLAST may be used in an iterated search to detect distant relationships between molecules (Id.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., for XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When aligning amino acid sequences using the ALIGN program, a PAM120 weight residue table can be used, with a gap length penalty of 12 and a gap penalty of 4.

[0164] In some embodiments, amino acid sequence modifications or alterations of the polypeptides described herein are contemplated. The alterations may be substitutions, deletions, or insertions of one or more codons encoding the polypeptide, resulting in an alteration of the amino acid sequence relative to the original polypeptide.

[0165] Amino acid substitutions may be the result of replacing an amino acid with another amino acid having similar structural and / or chemical properties, such as replacing leucine with serine, and may be, for example, conservative amino acid substitutions. Standard techniques well known to those of skill in the art may be used to introduce mutations into nucleotide sequences encoding the molecules herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Tolerable changes can be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity of the parent polypeptide.

[0166] The present disclosure includes polypeptides produced by conservative amino acid substitutions. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As noted above, the art has defined families of amino acid residues with side chains with similar charges. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, e.g., by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, and the activity of the protein can be determined. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to retain or not significantly alter properties.

[0167] Amino acids can be grouped based on the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry 73-75 (2nd ed., 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, to improve the oxidative stability of the molecule and prevent aberrant crosslinking, any cysteine ​​residue not involved in maintaining the correct conformation of the peptide may be substituted with another amino acid, such as alanine or serine. Non-conservative substitutions involve exchanging one member of these classes for another.

[0168] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Alterations may be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. To generate polypeptide variant DNA, cloned DNA may be subjected to site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques.

[0169] signal peptide In certain embodiments, a chimeric cytokine receptor according to the present specification may comprise a signal peptide (also referred to as a signal sequence) at the N-terminal position of the polypeptide. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of a cell and enables incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides applicable to the CARs described herein, including signal sequences of naturally occurring proteins or synthetic non-natural signal sequences, will be apparent to those skilled in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0170] In another aspect, the description provides a polynucleotide, the polynucleotide encoding a chimeric cytokine receptor according to the description. In another aspect, the description provides an immune cell, the immune cell expressing a chimeric cytokine receptor according to the description.

[0171] Tag peptide In certain embodiments, a chimeric cytokine receptor according to the present disclosure may comprise a tag. In some embodiments, a chimeric cytokine receptor may comprise two or more tags. In some embodiments, the tag linked to the chimeric cytokine receptor is different from the tag linked to the functional exogenous receptor.

[0172] 5.2.2. Functional exogenous receptors In some embodiments, the immune effector cells according to the present disclosure further comprise one or more functional exogenous receptors. In some embodiments, the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen conjugate (TAC)-like chimeric receptor. In some embodiments, the functional exogenous receptor is a CAR. In other embodiments, the functional exogenous receptor is a TCR.

[0173] Any functional exogenous receptor is encompassed by the present disclosure. Chimeric antigen receptors (CARs) are described in more detail below as exemplary functional exogenous receptors according to the present disclosure, but are not intended to limit the scope of the present disclosure.

[0174] In some embodiments, the CAR in the immune effector cells of the invention comprises a polypeptide, wherein the polypeptide comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0175] Extracellular antigen-binding domain The extracellular antigen-binding domain of a CAR described herein comprises one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain of a CAR described herein is monospecific. In other embodiments, the extracellular antigen-binding domain of a CAR described herein is multispecific. In other embodiments, the extracellular antigen-binding domain of a CAR described herein is monovalent. In other embodiments, the extracellular antigen-binding domain of a CAR described herein is multivalent. In some embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains, which are fused to each other by a direct peptide bond or via a peptide linker. In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof, as described in the context of the extracellular domain of a chimeric cytokine receptor in Section 5.2.1, above. When multiple binding domains are present in the extracellular antigen-binding domain of a CAR of the invention, the various domains can be fused to each other via peptide linkers. In some embodiments, the domains are fused to each other directly without any peptide linker. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence, depending on the structural and / or functional characteristics of the various domains. Each peptide linker may be independently selected and optimized. The length, flexibility, and / or other properties of one or more peptide linkers used in a CAR may have some effect on properties, including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. In some embodiments, the peptide linker contains flexible residues (e.g., glycine and serine), allowing adjacent domains to move freely relative to one another. For example, a glycine-serine pair may be a suitable peptide linker. Section 5.2.1 above provided further description of suitable linkers for use herein.

[0176] In some embodiments, the extracellular antigen-binding domain of the CAR of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. Tumors express proteins that can act as target antigens for immune responses (particularly T cell-mediated immune responses). The antigen targeted by the CAR may be an antigen in a single diseased cell or an antigen expressed in different cells that each affect the disease. The antigen targeted by the CAR may be directly or indirectly involved in the disease.

[0177] In some embodiments, Section 5.2.1 above provided antigens that can be targeted by the CARs provided herein. In some embodiments, the target cell is a cancer cell. In one embodiment, the target of the CAR of the present invention is a pathogen. In a specific embodiment, the target cell is a cell containing a pathogen. In some embodiments, the pathogen causes an infectious disease. In some embodiments, the pathogen is a bacterium. In some embodiments, the pathogen is a parasite. In some embodiments, the pathogen is a virus.

[0178] Transmembrane domain The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused with an extracellular antigen-binding domain. The transmembrane domain can be derived from natural or synthetic sources. The transmembrane domain suitable for the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-natural protein segment, such as a hydrophobin segment that is thermodynamically stable in the cell membrane.

[0179] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, a transmembrane domain from a multi-pass membrane protein may be compatible for use in a CAR described herein. A multi-pass membrane protein may comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helices or beta sheet structures. In some embodiments, the N- and C-termini of a multi-pass membrane protein are on opposite sides of a lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer, while the C-terminus of the protein is on the extracellular side.

[0180] The transmembrane domain used in the CARs described herein may further comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art, e.g., U.S. Pat. No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0181] A transmembrane domain according to the present specification may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, favors orientation of the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0182] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR according to the present specification comprises a hydrophobic artificial sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminal position of the transmembrane domain. In some embodiments, the transmembrane region comprises primarily hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a polyleucine-alanine sequence. The hydropathy or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydropathy analysis.

[0183] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0184] In some specific embodiments, the transmembrane domain is derived from CD8. In some particular embodiments, the transmembrane domain is derived from CD8α. In other particular embodiments, the transmembrane domain is derived from CD28.

[0185] Intracellular signaling domains The intracellular signaling domain in a CAR according to the present specification is responsible for activating at least one normal effector function of an immune effector cell expressing the CAR. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or accessory activity (including cytokine secretion). Thus, the term "intracellular signaling domain" refers to a protein portion that transmits an effector function signal to instruct a cell to perform a specific function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. Regarding the use of truncated portions of the intracellular signaling domain, such truncated portions may be used in place of the intact chain, so long as they transmit the effector function signal. Thus, the term intracellular signaling domain is intended to refer to any truncated portion that includes sufficient intracellular signaling domain to transmit the effector function signal.

[0186] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, a CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to an intracytoplasmic signaling sequence that stimulates and induces immune effectors to function. In some embodiments, the primary intracellular signaling domain contains a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. As used herein, "ITAM" is a conserved protein motif typically found in the tails of signaling molecules expressed in immune cells. The motif may comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs in signaling molecules are important for intracellular signal transduction, which is mediated, at least in part, by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G), FcR beta (Fc ε Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0187] Costimulatory signaling domain In some embodiments, the CAR comprises at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signaling to induce an immune response (e.g., effector function). In addition to stimulating antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival, as well as to activate cellular effector functions.

[0188] The costimulatory signaling domain of the chimeric receptor described herein can be an intracellular signaling domain from a costimulatory protein, which transmits a signal to regulate responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A "costimulatory signaling domain" can also be the intracytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a homologous binding partner in an immune cell (e.g., a T cell) that mediates a costimulatory response of the immune cell, including, but not limited to, proliferation and survival, by specifically binding to a costimulatory ligand.

[0189] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., about any one of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins (e.g., any two or more costimulatory signaling domains described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3z) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3z) are fused to each other via any peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3z). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0190] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be compatible for use in the CARs described herein. The type or types of costimulatory signaling domains are selected based on factors such as, for example, the type of immune effector cell on which the effector molecule is expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effector). Examples of costimulatory signaling domains for use in CARs include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6), and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF), and CAR-specific co-stimulatory domains (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9 ... R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNFRII / TNFRSF1B), SLAM family members (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150), and any other costimulatory molecules, e.g., CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA The intracellular signaling domains may be intracellular signaling domains of costimulatory proteins, including, but not limited to, class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of ligands that specifically bind to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0191] In some embodiments, the costimulatory signaling domain is a variant of any costimulatory signaling domain described herein, such that the costimulatory signaling domain can modulate the immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue mutations compared to a wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid changes may be referred to as variants. Compared to a costimulatory signaling domain that does not comprise a mutation, mutations in amino acid residues in the costimulatory signaling domain can result in increased signaling and enhanced stimulation of the immune response. Compared to a costimulatory signaling domain that does not comprise a mutation, mutations in amino acid residues in the costimulatory signaling domain can result in decreased signaling and reduced stimulation of the immune response.

[0192] signal peptide In certain embodiments, a CAR according to the present disclosure may comprise a signal peptide (also referred to as a signal sequence) at the N-terminal position of the polypeptide. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of a cell and enables incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic, non-natural signal sequences, that are compatible for use in the CARs described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0193] Hinge Area In some embodiments, a CAR according to the present disclosure may comprise a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is typically expressed between two domains of a protein and can allow one or both flexible domains of the protein to move relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0194] The hinge domain of an antibody (e.g., an IgG, IgA, IgM, IgE, or IgD antibody) is also applicable to the pH-dependent chimeric receptor system described herein. In some embodiments, the hinge domain is the hinge domain connecting the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is the hinge domain of an antibody and comprises the hinge domain of an antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the hinge domain of the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the hinge domain of the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0195] Non-naturally occurring peptides may be used as the hinge domain of the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker, such as a (GxS)n linker, where x and n are independently integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0196] The hinge domain may comprise any one of about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the length of the hinge domain may be at least any one of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0197] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Any hinge domain of a protein known in the art that contains a hinge domain is applicable to the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor.

[0198] In some specific embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., comprises a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In other embodiments, the hinge domain is derived from CD28α. In some embodiments, the hinge domain is a portion of the hinge domain of CD28α, e.g., comprises a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD28α.

[0199] Tag peptide In some embodiments, the functional exogenous receptor may comprise a tag. In some embodiments, the functional exogenous receptor may comprise two or more tags. In some embodiments, the tag linked to the chimeric cytokine receptor is different from the tag linked to the functional exogenous receptor.

[0200] Exemplary CAR Any CAR can be used in the present disclosure, including but not limited to the CARs in this disclosure.

[0201] In certain embodiments, a CAR according to the present specification comprises an amino acid sequence having a certain percent identity to any one of the exemplary CARs described above and infra in Section 6. In some embodiments, the present specification provides a CAR comprising or consisting of an extracellular domain, which has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a CAR described above and infra in Section 6.

[0202] In some embodiments, modifications of one or more amino acid sequences of the CARs described herein are contemplated. For example, optimization of the binding affinity and / or other biological properties of the extracellular domain, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility, may be required. Therefore, in addition to the extracellular domains described herein, it is also anticipated that variants of the domains described herein may be produced. For example, scFv variants can be produced by introducing appropriate nucleotide changes into the encoding DNA and / or synthesizing the desired antibody or polypeptide. It will be understood by those skilled in the art that amino acid modifications may alter post-translational processing of the antibody.

[0203] The alterations may be the substitution, deletion, or insertion of one or more codons encoding the polypeptide, resulting in an alteration of the amino acid sequence relative to the original antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0204] Amino acid substitutions may result from replacing an amino acid with another amino acid having similar structural and / or chemical properties, such as replacing leucine with serine, and may be, for example, conservative amino acid substitutions. Standard techniques well known to those of skill in the art may be used to introduce mutations into nucleotide sequences encoding molecules herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Tolerable changes can be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity of the parent polypeptide.

[0205] The present disclosure includes polypeptides produced by conservative amino acid substitutions. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) may be made to retain or not significantly alter properties.

[0206] One type of substitutional variant involves substituting one or more hypervariable region residues or fragments thereof of a parent antibody (e.g., a humanized or human antibody) in the extracellular antigen-binding domain of a CAR of the invention. Generally, the resulting variant selected for further study has an alteration (e.g., improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retains a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be readily generated using, for example, phage-display-based affinity maturation techniques, as described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0207] Modifications (e.g., substitutions) can be made in CDRs to, for example, improve antibody affinity. Such modifications may be made in CDR "hotspots," i.e., residues encoded by codons frequently mutated during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), where the resulting variant antibodies or fragments thereof are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (ed. by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis), and a secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR orientation, in which a number of CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.

[0208] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions according to the present disclosure) may be made in a CDR that do not significantly reduce binding affinity. In some embodiments, each CDR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0209] As described in Cunningham and Wells, Science, 244:1081-1085 (1989), a useful method for identifying antibody residues or regions amenable to targeted mutagenesis is called "alanine scanning mutagenesis." Alternatively, or additionally, contact points between the antibody and antigen can be identified by a crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted as substitution candidates or removed. Variants can be screened to determine whether they contain the desired properties.

[0210] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusing the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antigen-binding domain antibody.

[0211] Changes may be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. To generate antibody variant DNA, cloned DNA may be subjected to site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques.

[0212] The present disclosure further includes immune effector cells that comprise two or more types of functional exogenous receptors, such as dual CARs or tandem CARs that bind to two different targets.

[0213] In some specific embodiments, the CAR described herein binds to phosphatidylinositol proteoglycan 3 (GPC3). GPC3 (a 65-kDa protein consisting of 580 amino acids) is a heparan sulfate proteoglycan anchored to the cell membrane via glycosylphosphatidylinositol. GPC3 is expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma (OCCC), melanoma, lung squamous cell carcinoma, hepatoblastoma, nephroblastoma (Wilms' tumor), and yolk sac tumor, as well as certain gastric cancers (e.g., alpha-fetoprotein-producing gastric cancer). Although the exact functions of secreted and membrane-anchored GPC3 in these cancers have not yet been fully elucidated, they have been shown to be associated with tumor transformation, for example, in HCC (Shirakawa H. et al., Cancer Sci. 2009;100:1403-1407). Notably, the protein is largely absent in all other forms of cancer. GPC3 sequences are known in the art. In some embodiments, the GPC3 comprises an amino acid sequence selected from at least one of GenBank accession numbers NM_001164617 and NP_001158089, NM_004484 and NP_004475, NM_001164618 and NP_001158090, and NM_001164619 and NP_001158091. In some specific embodiments, a CAR that binds to GPC3 according to the present disclosure comprises the amino acid sequence of SEQ ID NO: 29 or 135.

[0214] 5.2.3. Immune effector cells "Immune effector cell" refers to an immune cell capable of exerting immune effector function. In some embodiments, the immune effector cell expresses at least FcγRIII and exerts ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0215] In some embodiments, the immune effector cells are T cells. In some embodiments, the T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + , CD4 - / CD8 - or a combination thereof. In some embodiments, the T cells express the CAR and produce IL-2, IFN, and / or TNF after binding to the target cell. In some embodiments, the CD8 + After expressing the CAR and binding to the target cell, the T cell lyses the antigen-specific target cell.

[0216] In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells may be an established cell line, such as NK-92 cells.

[0217] In some embodiments, immune effector cells are differentiated from stem cells (eg, hematopoietic stem cells, pluripotent stem cells, iPS, or embryonic stem cells).

[0218] The engineered immune effector cells are produced by introducing a polypeptide according to the present disclosure into an immune effector cell, such as a T cell. In some embodiments, the polypeptide is introduced into the immune effector cell by transfecting it with any one of the isolated nucleic acids or any one of the vectors described above.

[0219] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical, or biological methods.

[0220] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, vectors are introduced into cells by electroporation.

[0221] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells.

[0222] Chemical methods for introducing vectors into immune effector cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0223] In some embodiments, RNA molecules encoding any of the polypeptides described herein can be produced by conventional methods (e.g., in vitro transcription) and introduced into immune effector cells by known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035 (2006).

[0224] In some embodiments, the transduced or transfected immune effector cells are expanded ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured and expanded for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select engineered mammalian cells.

[0225] Reporter genes can be used to identify transfectable cells and evaluate the function of regulatory sequences. Typically, reporter genes are genes that are absent or not expressed in recipient organisms or tissues, and they encode polypeptides whose expression is indicated by some easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is detected at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0226] Other methods for confirming the presence of nucleic acids encoding polypeptides in engineered immune effector cells include molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and biochemical assays that detect the presence or absence of specific peptides, for example, by immunological methods (e.g., ELISA and Western blot).

[0227] 5.2.4. Source of T cells In some embodiments, a source of T cells is obtained from a subject prior to expansion and genetic modification of the T cells. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, multiple T cell lines available in the art can be used. In some embodiments, multiple T cell lines can be obtained using multiple techniques well known to those of skill in the art (e.g., Ficoll). (商標) T cells can be obtained from a unit of blood collected from a subject using apheresis (isolation). In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis are washed to remove the plasma portion, and the cells may be used for subsequent processing steps in an appropriate buffer or medium. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many (but not all) divalent cations. An initial activation step in the absence of calcium can lead to expanded activation. As one of ordinary skill in the art will readily appreciate, wash steps can be completed by methods known in the art, such as using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, e.g., Ca 2+ None, Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as PBS without buffer, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0228] In some embodiments, for example, PERCOLL (商標)T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes by gradient centrifugation or counterflow centrifugal elutriation. CD3 + , CD28 + , CD4+, CD8 + , CD45RA + and CD45RO + Specific subgroups of T cells, such as T cells, can be further isolated. For example, in some embodiments, T cells are cultured on anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads (e.g., DYNABEADS) for a sufficient time to positively select for the desired T cells. (登録商標) T cells are isolated by incubation with CD8 T cells (M-450 CD3 / CD28 T cells). In some embodiments, the period is about 30 minutes. In other embodiments, the period is 30 minutes to 36 hours or more, and all integer values ​​therebetween. In other embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For the isolation of T cells from leukemia patients, longer incubation times (e.g., 24 hours) can increase cell yield. Longer incubation times can be used to isolate T cells in any case where T cells are scarce relative to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Longer incubation times can also be used to isolate T cells. It should be noted that longer incubation times can also increase the number of CD8 T cells. +This can improve the efficiency of T cell capture. Thus, in some embodiments, subpopulations of T cells can be preferentially selected for or deselected at the initiation of culture or at other time points during culture by simply shortening or lengthening the time allowed for binding of T cells to CD3 / CD28 beads and / or increasing or decreasing the bead to T cell ratio. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or deselected at the initiation of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used. In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells for the activation and expansion process. "Unselected" cells can also undergo additional rounds of selection.

[0229] Enrichment of T cell populations by negative selection can be achieved by binding with antibodies directed against surface markers unique to the negatively selected cells. One method involves cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, CD4 + , CD25+, CD62Lhi, GITR + and FoxP3 + Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0230] The concentration of cells and surfaces (e.g., particles (e.g., beads)) may be varied to isolate the desired cell population by positive or negative selection. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase cell expansion) to ensure maximum cell-to-bead contact. For example, in one embodiment, a concentration of 2 billion cells / mL is used. In one embodiment, a concentration of 1 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / mL is used. In other embodiments, concentrations of 125 million or 150 million cells / mL may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations may enable more effective capture of cells that may weakly express the target antigen of interest (e.g., CD28-negative T cells) or cells from samples containing many tumor cells (e.g., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be readily available. In some embodiments, the use of higher cell concentrations allows for more effective selection of CD8+ T cells, which typically have weak CD28 expression.

[0231] In some embodiments, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of the desired antigen for binding to the particles. For example, CD4 + T cells express higher levels of CD28 and are captured more effectively than dilute concentrations of CD8+ T cells. In some embodiments, the concentration of cells used is 5×10 6In some embodiments, the concentration used is about 1 x 10 5 pieces / mL~1×10 6 The value can be any integer value between 0.01 and 0.15, and can be any integer value between 0.01 and 0.15.

[0232] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at 2° C.-10° C. or at room temperature.

[0233] T cells for stimulation may be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing steps may provide a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in such cases, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or medium containing 31.25% plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and freezing the cells to -80°C at a rate of 1°C / min and storing them in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods and uncontrolled immediate freezing at -20°C or liquid nitrogen can be used.

[0234] In some embodiments, cryopreserved cells are thawed, washed, and allowed to stand at room temperature for one hour before activation, as described herein.

[0235] The present disclosure also contemplates collecting a blood sample or apheresis product from a subject at a time prior to the potential need for the expanded cells described herein. Thus, a cell source for expansion can be collected at any desired time, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy, such as for multiple diseases or conditions that would benefit from T cell therapy, as described herein. In one embodiment, the blood sample or apheresis blood component is obtained from a normal, healthy subject. In certain embodiments, the blood sample or apheresis blood component is obtained from a normal, healthy subject who is at risk for, but does not have, a disease, and the desired cells are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen, and later used. In certain embodiments, the sample is obtained from a patient shortly after diagnosis of a particular disease, as described herein, but prior to any treatment. In further embodiments, cells are isolated from a subject's blood sample or apheresis blood component prior to multiple associated therapeutic modalities, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies or other immunoablative agents (e.g., CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228), and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Liu et al., Cell 66:807-815 (1991); Henderson et al., Immun 73:316-321 (1991); Bierer et al., Curr. Opin. Immun. 5:763-773 (1993)).In a further embodiment, the patient's cells are isolated and frozen for later use in conjunction with (e.g., before, concurrently with, or after) bone marrow or stem cell transplant, T cell ablative therapy using chemotherapeutic agents (e.g., fludarabine), external beam radiation therapy (XRT), cytoxan, or antibodies (e.g., OKT3 or CAMPATH). In another embodiment, the cells may be isolated before B cell ablative therapy (e.g., an agent reactive with CD20, such as Rituxan) and frozen for use in subsequent treatment.

[0236] In some embodiments, T cells are obtained during treatment or directly from a patient. In this regard, it has been observed that after certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the obtained T cells may be optimal or their ex vivo expansion capacity may be improved immediately after treatment, during the period when the patient would normally be recovering from treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, in the context of the present disclosure, it is contemplated to harvest blood cells, including T cells, dendritic cells, or other hematopoietic cells, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used in a subject to create conditions favorable for the repletion, recirculation, regeneration, and / or expansion of cell types, particularly for a defined time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0237] 5.2.5. T cell activation and expansion In some embodiments, before or after genetic modification of the T cells with a CAR described herein, the T cells can generally be activated and expanded using methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0238] Typically, T cells can be expanded by contacting them with a surface to which is attached an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, as described herein, T cell populations can be stimulated by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by a protein kinase C activator (e.g., bryostatin) bound to a calcium ion vector. Ligands that bind to helper molecules can also be used to costimulate helper molecules on the T cell surface. For example, T cell populations can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. CD4 + T cells or CD8 +Anti-CD3 and anti-CD28 antibodies may be used to stimulate T cell proliferation. Examples of anti-CD3 antibodies include UCHT1, OKT3, and HIT3a (BioLegend, San Diego, US), and may be used as well as other methods known in the art (Graves J et al., J. Immunol. 146:2102 (1991); Li B et al., Immunology 116:487 (2005); Rivollier A et al., Blood 104:4029 (2004)). Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), as well as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, (1998); Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63 (1999)).

[0239] In some embodiments, the primary and costimulatory signals for T cells can be provided by various regimens. For example, the agents providing each signal can be in solution or coupled to a surface. If coupled to a surface, the agents can be coupled to the same surface (i.e., in a "cis" configuration) or to a separate surface (i.e., in a "trans" configuration). Alternatively, one agent can be coupled to a surface and another agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In other embodiments, the agents can be in soluble form and cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which describe artificial antigen-presenting cells (aAPCs), which are contemplated for use in certain embodiments of the present disclosure for T cell activation and expansion.

[0240] In some embodiments, T cells are combined with agent-coated beads, followed by separation of the beads and cells and culturing the cells. In alternative embodiments, the agent-coated beads and cells are not separated prior to culturing, but are cultured together. In further embodiments, the beads and cells are first concentrated by applying a force, such as a magnetic force, to increase ligation of cell surface markers, thereby inducing cell stimulation.

[0241] For example, cell surface proteins can be linked by allowing T cells to contact paramagnetic beads (3 x 28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 4 ~4×10 8T cells) and beads (e.g., anti-CD3 / CD28 MACSiBead particles with a recommended titer of 1:100) are combined in a buffer solution, preferably PBS (without divalent cations such as calcium and magnesium). One of skill in the art will readily appreciate that any cell concentration can be used. For example, the target cells can be present in very small amounts in the sample, accounting for as little as 0.01% of the sample, or the entire sample (i.e., 100%) can contain the desired target cells. Thus, any cell number is within the scope of the present disclosure. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase cell expansion) to ensure maximum cell-particle contact. For example, in one embodiment, a concentration of approximately 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL are used. In yet another embodiment, cell concentrations of 75, 80, 85, 90, 95, or 100 million cells / mL are used. In further embodiments, concentrations of 125 or 150 million cells / mL may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations may enable more effective capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are expected to be available in certain embodiments. For example, the use of higher cell concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations may result in more effective capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are expected to be available in certain embodiments. For example, the use of higher cell concentrations may result in increased cell yield, cell activation, and cell proliferation. + Allows for more effective selection of T cells.

[0242] In some embodiments, the mixture can be cultured for any integer time between a few hours (about 3 hours) and about 14 days, or both. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several stimulation cycles may be necessary, as T cell culture times can be 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors essential for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may contain RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, X-Vivo 20, or optimizer supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma), a defined set of hormones, and / or a certain amount of cytokines sufficient to grow and expand T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and are not included in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to different stimulation times may exhibit various characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product contains more helper T cell populations (TH, CD4) than cytotoxic or suppressor T cell populations (TC, CD8). +). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population that is primarily composed of TH cells before about day 8-9, and after about day 8-9, the T cell population includes an increasingly abundant TC cell population. Thus, for therapeutic purposes, it may be advantageous to infuse a T cell population that is primarily composed of TH cells into a subject. Similarly, if an antigen-specific subpopulation of TC cells is isolated, it may be beneficial to expand that subpopulation to a greater extent.

[0243] It should be noted that in addition to CD4 and CD8 markers, other phenotypic markers also vary significantly but are, for the most part, reproducible during cell expansion, allowing for the customization of activated T cell products for specific purposes.

[0244] 5.3. Polypeptides Comprising Functional Exogenous Receptors and Chimeric Cytokine Receptors In another aspect, the present specification provides polypeptides, which comprise at least one functional exogenous receptor (e.g., a CAR, a TCR, or a TAC) and a chimeric cytokine receptor according to the present specification. The chimeric cytokine receptor in the polypeptides of the present invention can be any one of those described above in Section 5.2.1. The functional exogenous receptor in the polypeptides of the present invention can be any one of those described above in Section 5.2.2.

[0245] In the polypeptides of the present invention, the chimeric cytokine receptor may be located at the N-terminus of the functional exogenous receptor, or the chimeric cytokine receptor may be located at the C-terminus of the functional exogenous receptor.

[0246] In some embodiments of the various polypeptides herein, the chimeric cytokine receptor and the functional exogenous receptor are linked to one another via a peptide linker, hi some embodiments, the peptide linker is a self-cleaving peptide, such as the 2A self-cleaving peptide, such that the chimeric cytokine receptor and the functional exogenous receptor become a single polypeptide after cleavage within the cell.

[0247] Members of the 2A peptide family are named after the viruses that first described them. For example, the first described 2A peptide, F2A, is derived from the foot-and-mouth disease virus. Self-cleaving 2A peptides, 18–22 amino acids in length, mediate "ribosomal skipping" between proline and glycine residues, inhibiting peptide bond formation without affecting downstream translation. These peptides enable multiple proteins to be encoded as polyproteins, which dissociate during translation into their component proteins. Self-cleaving peptides are present in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), zosea azygna virus (TaV), and porcine teschovirus-1 (PTV-1) (see Donnelly et al., J. Gen. Virol., 82: 1027-101 (2001); Ryan et al., J. Gen. Virol., 72: 2727-2732 (2001)), and cardioviruses such as Theiloviruses (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively, and are included in the present disclosure, as described in, for example, Donnelly et al., J. Gen. Virol., 78: 13-21 (1997), Ryan and Drew, EMBO J., 13: 928-933 (1994), Szymczak et al., Nature Biotech., 5: 589-594 (2004), and Hasegawa et al., Stem Cells, 25(7): 1707-12 (2007). In other embodiments, an intein-mediated protein splicing system is used, as described herein in Shah and Muir, Chem Sci., 5(1): 446-461 (2014) and Topilina and Mills, Mobile DNA, 5 (5) (2014).Other methods known in the art can also be used to construct the present invention.

[0248] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide P2A fragment. In a specific embodiment, the self-cleaving peptide is a T2A fragment.

[0249] In another aspect, the description provides nucleic acids encoding the above-described polypeptides, which include functional exogenous receptors (e.g., CARs) according to the description, and functional exogenous receptors, which are described in more detail below.

[0250] Polynucleotides In another aspect, the disclosure provides polynucleotides encoding polypeptides according to the present invention, including those described in Sections 5.2 and 5.3 above.

[0251] More specifically, in some embodiments, the description provides a polynucleotide encoding a polypeptide, the polypeptide comprising a chimeric antigen receptor and a functional exogenous receptor (e.g., a CAR), wherein the chimeric cytokine receptor and the functional exogenous receptor are linked to each other via a peptide linker (e.g., a 2A self-cleaving peptide linker).

[0252] In yet another aspect, the present disclosure provides a polynucleotide comprising a region encoding a chimeric cytokine receptor according to the present disclosure (e.g., as described in Section 5.2.1) and a region encoding a functional exogenous receptor according to the present disclosure (e.g., as described in Section 5.2.2). In some embodiments, the two regions are controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, the regions are controlled by a single promoter.

[0253] In yet another aspect, the present specification provides a composition comprising a first polynucleotide encoding a chimeric cytokine receptor in accordance with the present specification (e.g., as described in Section 5.2.1) and a second polynucleotide encoding a functional exogenous receptor in accordance with the present specification (e.g., as described in Section 5.2.2).

[0254] The polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0255] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of the polypeptides of the present disclosure. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a polypeptide of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide has identity to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with alternative nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These variations in the reference sequence may occur anywhere at or between the 5' or 3' terminal positions of the reference nucleotide sequence, interspersed among single nucleotides in the reference sequence, or in one or more contiguous groups within the reference sequence.

[0256] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions, which do not result in a change in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be produced for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing the codons of human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0257] In some embodiments, polynucleotide variants are generated to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease expression of the polypeptide. In some embodiments, polynucleotide variants exhibit increased expression of the encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants exhibit decreased expression of the encoded polypeptide compared to the parent polynucleotide sequence.

[0258] Vectors Further provided are vectors comprising the polynucleotides or nucleic acid molecules described herein. In one embodiment, the nucleic acid molecules may be incorporated into a recombinant expression vector.

[0259] The present disclosure provides vectors for cloning and expressing any one of the polypeptides described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, cowpox vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0260] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, reverse-transcription viruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into reverse-transcription viral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many reverse-transcription viral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using packaging regimens known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art. Vectors derived from reverse-transcription viruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term and stable integration of transgenic vectors and propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0261] In some embodiments, a vector comprises any one of the nucleic acids encoding a polypeptide described herein. The nucleic acid can be cloned into the vector using any molecular cloning method known in the art, including, for example, using a restriction endonuclease site and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulatable promoters, such as inducible promoters.

[0262] In some embodiments, the nucleic acid encoding the polypeptide is operably linked to a constitutive promoter. A constitutive promoter allows for constitutive expression of a heterologous gene (also called a transgenic gene) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the murine stem cell virus (MSCV) promoter, the cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (hEF1α), the ubiquitin C promoter (UbiC), the phosphoglycerokinase promoter (PGK), the simian virus 40 early promoter (SV40), and the chicken β-actin promoter (CAGG) coupled to the CMV early enhancer. The efficiency of such constitutive promoters for driving transgenic expression has been extensively compared in many studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK to drive chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only induces the highest levels of transgenic expression but is also optimally maintained in CD4 and CD8 human T cells (Molecular Therapy, 17(8): 1453-1464 (2009)). In some embodiments, the nucleic acid encoding the CAR is operably linked to the hEF1α promoter. In some embodiments, the nucleic acid encoding the CAR is operably linked to the MSCV promoter.

[0263] In some embodiments, the nucleic acid encoding the polypeptide is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the engineered immune effector cells or the physiological state of the engineered immune effector cells, an inducer (i.e., an inducer agent), or a combination thereof.

[0264] In some embodiments, the inducing conditions do not induce expression of endogenous genes in the engineered mammalian cell and / or the subject receiving the pharmaceutical composition, hi some embodiments, the inducing conditions are selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and an activation state of the engineered mammalian cell.

[0265] In some embodiments, the vector contains a selectable marker gene or reporter gene to select cells expressing the polypeptide from a group of host cells transfected with the lentiviral vector. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cells. For example, the vector contains transcription and translation terminators, initiation sequences, and promoters that can be used to regulate expression of the nucleic acid sequence.

[0266] 5.6. Manufacturing method In yet another aspect, the description provides a method for producing an engineered immune effector cell (e.g., a CAR-T cell or a TCR-T cell or a TAC-T cell), the method comprising introducing one or more polynucleotides or one or more vectors according to the description (e.g., as described in Sections 5.4 and 5.5 above) into an immune effector cell (e.g., a T cell).

[0267] More specifically, in some embodiments, engineered immune effector cells according to the present disclosure can be produced by introducing into T cells one or more nucleic acids encoding a polypeptide described in part 5.3 or one or more nucleic acids described in section 5.4.

[0268] The chimeric cytokine receptor and the functional exogenous receptor can each be introduced into a T cell alone as a single polypeptide.

[0269] Alternatively, engineered immune effector cells according to the present disclosure can be produced from a polynucleotide containing multiple regions, such as a region encoding a CAR and a region encoding a chimeric cytokine receptor. The different regions can be controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, the different regions are controlled by a single promoter.

[0270] In yet another aspect, the description provides engineered immune effector cells (e.g., CAR-T cells) produced according to the methods herein.

[0271] The methods described above in the context of CAR-T cells are also applicable to the production of other immune effector cells (e.g., TCR-T cells and TAC-T cells).

[0272] 5.7. Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising the engineered cells of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the engineered T cells of the present disclosure and a pharmaceutically acceptable excipient.

[0273] In specific embodiments, the term "excipient" may further refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), carrier, or vehicle. Pharmaceutical excipients may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline, aqueous dextrose, and glycerin solutions can also be used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions, if desired, may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredients provided herein, e.g., in purified form, and a suitable amount of excipients to provide a form suitable for administration to a patient. The formulation should suit the mode of administration.

[0274] In some embodiments, the choice of excipient is determined in part by the particular cells and / or the mode of administration, and as such, multiple suitable formulations exist.

[0275] Typically, acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite, preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes), chelating agents such as EDTA, and / or non-ionic surfactants.

[0276] Buffers can be used to control the pH within a range that optimizes therapeutic efficacy, especially when stability is pH-dependent. Buffers suitable for the present disclosure include organic acids, inorganic acids, and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may include histidine and trimethylamine salts, such as Tris.

[0277] Preservatives may be added to retard microbial growth. Suitable preservatives for the present disclosure include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium halides (e.g., chlorine, bromine, iodine), benzethonium chloride, thimerosal, phenol, butyl or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0278] Tonicity agents (sometimes called "stabilizers") may be present to adjust or maintain the fluid tension in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often called "stabilizers" because they can reduce the likelihood of inter- and intramolecular interactions by interacting with the charged groups on the amino acid side chains. Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0279] Other exemplary excipients include (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as listed above), amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like, sugars such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycols, and the like. These include organic sugars or sugar alcohols such as ethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; human serum albumin, bovine serum albumin, gelatin, or other immunoglobulin low molecular weight proteins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0280] A non-ionic surfactant or detergent (also called a "wetting agent") may be present to promote dissolution of the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Available anionic detergents include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0281] The route of administration is in accordance with known and acceptable methods, such as single or multiple bolus injections or injection or infusion by any suitable route, e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular, topical administration, inhalation, or prolonged infusion by sustained or delayed release modes.

[0282] In another embodiment, the pharmaceutical composition can be provided as a controlled-release or sustained-release system. In one embodiment, a pump can be used to achieve controlled-release or sustained-release (see, e.g., Sefton, Crit. Ref. Biomed.Eng. 14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. J. Med. 321:569-74 (1989)). In another embodiment, polymeric materials may be used to provide controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein described herein) or composition herein (see, e.g., Medical Applications of Controlled Release (eds. Langer and Wise, 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (eds. Smolen and Ball, 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12 (1989), U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326, and PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253.) Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, stable during storage, sterile, and biodegradable. In yet another embodiment, a controlled-release or sustained-release system may be placed near a specific target tissue, such as the nasal passages or lungs, so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to one of skill in the art may be used to prepare sustained-release formulations containing one or more of the agents described herein (see, e.g., U.S. Pat. No. 4,526,938; PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Song et al., PDA J. of Pharma.Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0283] The pharmaceutical compositions described herein may further comprise two or more active compounds or agents as necessary for the particular indication being treated. Alternatively, or additionally, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, or growth-inhibitory agent. Such molecules are present in any suitable combination in amounts effective for the desired purpose.

[0284] The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th Edition.

[0285] A variety of compositions and delivery systems are known and can be used with the therapeutic agents according to the present invention, including nucleic acids encapsulated in liposomes, microparticles, microcapsules, recombinant cells, retroviral vectors or other vectors capable of expressing the single domain antibodies or therapeutic molecules according to the present invention.

[0286] In some embodiments, pharmaceutical compositions herein contain binding molecules and / or cells in an amount effective to treat or prevent a disease or disorder, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodic evaluation of the treated subject. For repeated administration over several days or more, treatment is repeated until a desired suppression of disease symptoms occurs, depending on the condition. However, other dosage regimens may be useful and can be determined.

[0287] 5.8. Methods and Uses The methods disclosed herein can be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides methods for treating cancer in a subject, for reducing the rate of increase in tumor volume over time in a subject, for reducing the risk of developing metastasis, or for reducing the risk of developing further metastases in a subject. In some embodiments, treatment can halt, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0288] As used herein, in some embodiments, treatment herein delays the progression of a disease or disorder, e.g., slows, prevents, decelerates, arrests, stabilizes, inhibits, and / or slows the progression of a disease (e.g., cancer). This delay may have different lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, since the individual is not afflicted with the disease or disorder. For example, advanced cancer (e.g., the progression of metastasis) may be delayed. In other embodiments, the method or use herein prevents a disease or disorder.

[0289] In some embodiments, the cell therapy of the present invention includes therapy containing T cells, where the T cells express various CARs, TCRs, cTCRs, TACs, TAC-like chimeric receptors, CARs and CCRs, TCRs and CCRs, cTCRs and CCRs, TACs and CCRs, TAC-like chimeric receptors and CCRs, or combinations thereof, as described herein. In some embodiments, the CAR-T cell therapy of the present invention is for treating solid tumor cancers. In other embodiments, the CAR-T cells of the present invention are for treating hematological cancers.

[0290] In some embodiments, the subject has a GPC3-, AFP-, claudin 18.2-, CD19-, CD20-, CD22-, BCMA-, GD2-, DLL3-, MSLN-, CD30-, CLL1-, or CD33-positive cancer. In some embodiments, the subject has liver cancer (e.g., hepatocellular carcinoma), glioma, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, renal cancer, urothelial cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, and / or ovarian cancer. In some embodiments, the subject has squamous cell lung cancer or a solid tumor. In some embodiments, the subject has a CNS tumor, thyroid cancer, gastrointestinal cancer, skin cancer, sarcoma, genitourinary cancer, and / or germ cell tumor. GPC3-associated cancers can be found, for example, in Moek, Kirsten L. et al., The American Journal of Pathology 188.9 (2018): 1973-1981, which is incorporated herein by reference in its entirety.

[0291] In other embodiments, the disease or disorder is an autoimmune or inflammatory disease. In other embodiments, the disease or disorder is an infectious disease. In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of arthritis, colitis, psoriasis, severe asthma, and moderate to severe Crohn's disease.

[0292] In some embodiments, the disease or disorder is a hematological cancer such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma, transformed lymphoma, moderately differentiated lymphocytic lymphoma, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocellular lymphoma, diffuse small cleaved cell lymphoma (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma, mantle layer lymphoma. In a specific embodiment, the disease or disorder is selected from the group consisting of acute myelogenous lymphoma, low-grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloid leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia (Burkitt's lymphoma), acute biphenotypic leukemia, chronic myelogenous lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. In another specific embodiment, the disease or disorder is myelodysplastic syndrome (MDS). In another specific embodiment, the disease or disorder is acute myeloid leukemia (AML). In another specific embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another specific embodiment, the disease or disorder is multiple myeloma (MM).

[0293] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of carcinoma, adenocarcinoma, adrenocortical carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, colorectal carcinoma, ductal cell carcinoma, lung cancer, thyroid cancer, nasopharyngeal carcinoma, melanoma, non-melanoma skin cancer, liver cancer, and lung cancer.

[0294] In some embodiments, the disease or disorder is caused by a pathogen. In some embodiments, the pathogen causes an infectious disease. In some embodiments, the pathogen is a bacterium. In some embodiments, the pathogen is a parasite. In some embodiments, the pathogen is a virus.

[0295] In other embodiments, the disease or disorder is an immune or autoimmune disorder. In some embodiments, the disease or disorder is an inflammatory disease. Inflammation plays an important role in host defense and the progression of immune-mediated diseases. The inflammatory response is a response to injury (e.g., wounds, ischemia, and exogenous particles) and infection (e.g., bacterial or viral infection) and is initiated by a complex cascade of events involving chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., white cells). The inflammatory response is characterized by increased blood flow, increased capillary permeability, and a massive influx of phagocytes. These events result in swelling, redness, fever (varies in fever type), and pus formation at the wound or infected site.

[0296] Methods for administering cells for adoptive cell therapy are well known and are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238, U.S. Patent No. 4,690,915, Rosenberg, Nat Rev Clin Oncol. 8 (10):577-85 (2011), Themeli et al., Nat Biotechnol. 31(10):928-933 (2013), Tsukahara et al., Biochem Biophys Res Commun 438(1): 84-9 (2013), and Davila et al., PLoS ONE 8(4): e61338 (2013). These methods can be used in conjunction with the methods and compositions herein.

[0297] In some embodiments, cell therapy (e.g., adoptive T cell therapy) is performed by autologous transplantation, where cells are isolated and / or otherwise produced from the subject receiving cell therapy or a sample derived from such a subject. Thus, in some aspects, cells are derived from a subject in need of treatment and, after isolation and processing, are administered to the same subject. In other embodiments, cell therapy (e.g., adoptive T cell therapy) is performed by allogeneic transplantation, where cells are isolated and / or otherwise produced from a subject other than the subject receiving or ultimately receiving cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA type or supertype as the first subject.

[0298] In some embodiments, the subject to which the cells, cell populations, or compositions are administered is a primate, such as a human. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing toxicity outcomes.

[0299] The compositions herein can be administered in any suitable manner, for example, by injection, such as intravenous or subcutaneous injection. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, and, when localized treatment is required, intralesionally.

[0300] The amount of a prophylactic or therapeutic agent herein that will effectively prevent and / or treat a disease or condition can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The appropriate dosage of the binding molecule or cells for disease prevention or treatment may depend on the type of disease or disorder being treated, the type of binding molecule, the severity and course of the disease or disorder, whether the agent is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the drugs, and the judgment of the attending physician. In some embodiments, the compositions, molecules, and cells are suitably administered to the patient at one time or over a series of treatments. Multiple doses can be administered intermittently. An initial, higher loading dose may be administered, followed by one or more lower doses.

[0301] In the case of genetically engineered cells, in some embodiments, a subject may be administered about 1 million to about 100 billion cells and / or such amounts of cells per kilogram of body weight. In some embodiments, the pharmaceutical composition comprises any one of the engineered immune cells described herein, and the pharmaceutical composition is administered in an amount of at least about 10 per kg of body weight of the individual. 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 The cells may be administered in any one dose, which may vary depending on the particular attributes of the disease or disorder and / or the patient and / or other treatments.

[0302] In some embodiments, the pharmaceutical composition is administered once. In some embodiments, the pharmaceutical composition is administered multiple times (e.g., any one of 2, 3, 4, 5, 6, or more times). In some embodiments, the pharmaceutical composition is administered once or multiple times during a dosing cycle. A dosing cycle may be, for example, 1, 2, 3, 4, 5, or more weeks, or 1, 2, 3, 4, 5, or more months. Optimal dosages and treatment regimens for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for disease symptoms and adjusting treatment accordingly.

[0303] In some embodiments, a composition according to the present invention is administered as part of a combination therapy, such as administered simultaneously with another therapeutic intervention (e.g., another antibody or engineered cell or receptor or agent, e.g., a cytotoxic or therapeutic agent), or sequentially in any order.

[0304] In certain embodiments, after the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell population is measured by any one of several known methods. Evaluation parameters include specific binding of engineered or natural T cells or other immune cells to an antigen in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells can be further measured by assaying the expression and / or secretion of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing clinical outcomes, such as tumor burden or tumor burden reduction.

[0305] 5.9. Kits and Products Further provided are kits, unit doses, and articles of manufacture comprising any one of the engineered immune effector cells described herein. In some embodiments, kits are provided containing any one of the pharmaceutical compositions described herein, and preferably instructions therefor are provided.

[0306] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film (Mylar) or plastic bags), etc. The kits can optionally provide other components such as buffers and instructional information. Thus, the present application further provides articles of manufacture, which include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0307] The article of manufacture may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials (e.g., glass or plastic). Typically, the container holds a composition that effectively treats a disease or disorder (e.g., cancer) described herein and may have a sterile access point (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The label or packaging insert indicates that the composition is used to treat a particular condition in an individual. The label or packaging insert further includes instructions for administering the composition to an individual. The label may provide instructions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. A package insert refers to instructions typically included in commercial packaging for a therapeutic product, including information regarding the indications, uses, dosages, administration, contraindications, and / or warnings for the use of such a therapeutic product. Additionally, the product may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0308] The kit or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions, packaged in an amount sufficient for storage and use in a pharmacy (eg, hospital pharmacy and compounding pharmacy).

[0309] The present disclosure is generally disclosed herein using affirmative language in describing many embodiments. The present disclosure also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, regimens, procedures, assays or analyses, is completely or partially excluded. Thus, even if the present disclosure is generally not presented herein based on content not included in the disclosure, aspects not explicitly included in the present disclosure are still disclosed herein.

[0310] Although several embodiments of the present disclosure have been described, it should be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. As such, the following examples are intended to illustrate, but not limit, the scope of the disclosure as set forth in the claims. [Example]

[0311] 6. Working Example The following are descriptions of various methods and materials used in the research and are intended to provide those skilled in the art with a complete disclosure and description of how to make and use the present disclosure, but are not intended to limit the scope of what the inventors consider to be their disclosure, nor are they intended to be all experiments that have been performed or could be carried out. It is understood that exemplary descriptions written in the present tense need not be performed, and that these descriptions may be performed to generate data or the like relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy in numbers used (e.g., amounts, percentages, etc.), but experimental error and deviation should be taken into account.

[0312] 6.1. Example 1 - Plasmid Construction, Virus Production, and Titer Evaluation CAR T cells armed with different chimeric cytokine receptors were engineered, as shown in Figures 1A-1B, 2A-2B, and 3A-3B and SEQ ID NOs: 30-134, 136-140, 142, 144, 146, 148, 150, and 151. To generate viral particles containing polynucleic acids encoding any of the systems disclosed herein, a lentiviral packaging plasmid mixture containing pMDLg / pRRE (Addgene #11251), pRSV-Rev (Addgene #11253), and pMD2.G (Addgene #11259) and the PLVX-EF1A (containing the targeting system) vector were premixed with polyetherimide (PEI) at a pre-optimized ratio and incubated at room temperature for 5 minutes. The transfection mixture was added dropwise to 293-T cells and gently mixed. The transfected 293-T cells were incubated overnight at 37°C under 5% CO2. Twenty-four hours after transfection, the supernatant was collected and centrifuged at 500 g for 10 min at 4°C to remove any cell debris. The centrifuged supernatant was filtered through a 0.45 μm PES filter and centrifuged at ultra-high speed for virus concentration. After centrifugation, the supernatant was carefully discarded, and the virus precipitate was washed away with pre-chilled DPBS. The virus concentration was measured. The virus was aliquoted and stored at -80°C. The virus titer was measured by functional transduction in T cell lines.

[0313] Briefly, pLVX-Puro (Clontech #632164) was used to modify the lentiviral vector by replacing the original promoter with the human elongation factor 1α promoter (hEF1α) and removing the puromycin resistance gene with EcoRI and BamHI using GenScript. PLVX-EF1A was further subjected to the lentiviral packaging procedure as described above.

[0314] 6.2. Example 2 - T Cell Transduction and FACS Analysis of Transduced T Cells γδ T cells or αβ T cells were prepared by adding 5 μM zoledronate and 1000 IU / mL IL-2 to PBMCs and cultured for 14 days with periodic changes of medium supplemented with 1000 IU / mL IL-2. Alternatively, γδ T cells or αβ T cells were isolated from PBMCs or umbilical cord blood (UCB) and stimulated with anti-γδ TCR or anti-αβ TCR antibodies and anti-CD3 (OKT3), followed by incubation with K562-based artificial antigen-presenting cells (aAPCs) at a 1:2 ratio for at least 10 days.

[0315] PBMCs were isolated from leukocyte-reduced material by density centrifugation (lymphoprep) and cryopreserved. The PBMCs were resuscitated and activated with zoledronic acid (5 μM) in AIM-V cell culture medium supplemented with IL-2 (1000 IU / ml) and 5% human AB serum, and stored in a humidified chamber (37°C, 5% CO2). Forty-eight hours after activation, the cells were transduced with a lentiviral vector encoding the system described in Example 1 at an MOI of 5 in the presence of 5 pg / ml polybrene. The transduction procedure was repeated the following day, followed by replenishment with fresh medium containing IL-2 (1000 IU / ml) the day after transduction. The cells were cultured in AIM-V supplemented with IL-2 (1000 IU / ml) in a humidified chamber, with periodic medium changes depending on the pH for further expansion. Ten days after transduction, the cells were harvested and the total number, purity, and transduction efficiency were determined. Cells were further enriched using negative TCRγ / δ+ T cell or TCRαβ+ T cell isolation kits (Miltenyi Biotec) before future application or cryopreservation.

[0316] 6.3. Example 3 - Quantification of Transgenic Expression Three days after transduction and thereafter (usually days 3, 7, and 14), expression in cells of the line described in Example 1 was assessed by flow cytometry. Aliquots of cells were collected from the culture, washed, pelleted, and resuspended in antibody (eBioscience) diluted in PBS + 0.5% FBS at a dilution factor of 100, such that 50–100 μl was added to each sample. The resuspended cells were resuspended in approximately 50–100 μl of solution. The cells were incubated at 4°C for 30 minutes. The reactive dye eFluor 780 or SYTOX Blue reactive dye was then added according to the manufacturer's instructions. After incubation, the cells were washed twice in PBS, resuspended in 100–200 μl of PBS, and analyzed. The mean fluorescence of the line was quantified by flow cytometry.

[0317] As shown in Figures 35 and 36, compared to anti-GPC3 CAR αβ T cells, NKG2D CAR T cells showed lower proliferation and responsiveness due to suicide and friendly fire during culture.

[0318] For anti-GPC3 CAR-T staining, cells were stained with PE anti-DYKDDDDK tag antibody (Biolegend). All flow cytometry experiments were analyzed using FlowJo (Tree Star, Inc.).

[0319] Table 1 shows the positivity rates of virus-infected T cells expressing or armed with different CARs.

[0320] [Table 1]

[0321] 6.4. Example 4 - Quantification of Ligand Expression on Tumor Cell Lines Surface expression of NKG2D ligands on the HCC cell line huh7 was measured. Huh7 cells were stained with anti-MIC A / B antibody (Biolegend), anti-ULBP-1 antibody (R&D), anti-ULBP-3 antibody (R&D), anti-ULBP-2, 5, 6 antibody (R&D), or anti-CD155 antibody (Biolegend).

[0322] As shown in Figure 4, huh7 cells appropriately express NKG2D ligands, MIC A / B, and ULBP-2, 5, and 6.

[0323] 6.5. Example 5 - In Vitro Cytotoxicity Assay To rapidly evaluate the antitumor activity of CAR-T cells (γδ T cells or αβ T cells) in vitro, a cytotoxicity FACS assay was performed. On day 8 after transduction, the transduced T cells were harvested and transduced with target cells (GPC3). + The CAR-T cells were incubated with HCC cell lines (Huh7 and PLC-PRF-5) and GPC3-HCC cell line (SK-HEP-1) at an E / T ratio of 3:1, 1:1, or 0.3:1 (effector:CAR-T / target:Huh7, PLC-PRF-5, or SK-HEP-1) for 16 hours. CFSE staining of target cells was performed in advance. Untransduced T cells (UNT) from the same lot were used as a negative control. The mixed cells were harvested and subjected to 7-ADD staining. Cytotoxicity was calculated using the following equation: 7AAD + (E + T): 7-ADD% in E / T co-incubation, 7AAD + (T): 7-ADD% in target alone. All anti-GPC3 CAR T cells (γδ T cells or αβ T cells) showed potent and significant cytotoxicity against huh7 cells. Cytotoxicity was calculated as follows: % cytotoxicity = (7AAD + (E + T) - 7AAD + (T)) / (1 - 7AAD + (T)) × 100.

[0324] 6.6. Example 6 - Detection of IFN-γ, GM-CSF, and TNF-α Secretion by HTRF To measure effector T cell activation and proliferation, production of effector cytokines such as IFN-γ, GM-CSF, and TNF-α was measured. Supernatants from in vitro cytotoxicity assays were collected to assess cytokine release induced by CARs. HTRF assays for IFN-γ (Cisbio, Cat# 62HIFNGPEH), GM-CSF (Cisbio, Cat# 62HGMCSFPEH), and TNFα (Cisbio, Cat# 62HTNFAPEH) were performed according to the manufacturer's instructions.

[0325] All anti-GPC3 CAR T cells (γδ T cells or αβ T cells) showed potent cytotoxic activity against huh7 cells and released IFN-γ, GM-CSF, and TNF-α in response to huh7 cells. NKG2D chimeric cytokine receptor-armed CAR T cells (γδ T cells or αβ T cells) secreted TNF-α, IFN-γ, and GM-CSF at levels similar to those of naked CAR T cells.

[0326] 6.7. Example 7 - Long-Term Cytotoxicity Assay To evaluate the long-term killing effect of CAR T cells (γδ T cells or αβ T cells), a long-term co-culture assay was performed, which mimics the dynamic killing process in vivo. In the absence of exogenous cytokines (IL-2), transduced or untransduced T cells (1 × 10 5 cells / well) and tumor cell lines (huh7, Raji, or U937 cells, 1 × 10 5 T cells (cells / well) were co-cultured in 24-well plates at an E:T ratio of 1:1. After 2 or 3 days of co-culture, a portion of these cells was harvested and stained for CD3. For subsequent co-culture assays, the remaining T cells were then re-challenged with fresh huh7 cells at the same E:T ratio. Co-culture was continued until tumor cells outgrowth. The T cell proliferation rate at each time point was calculated by dividing the number of T cells at that time point by the number of T cells at the starting time point.

[0327] Figures 5, 7, 9, 11, 13, and 15 show the long-term cytotoxicity of BM CAR γδ T cells or αβ T cells armed with NKG2D chimeric single or dual cytokine receptors as detected by FACS. Figures 6, 8, 10, 12, 14, and 16 show calculated T cell proliferation from the same experiments. The data show that NKG2D chimeric cytokine receptor arming improved the cytotoxicity and proliferation of BM CAR T cells.

[0328] Figure 17 shows the long-term cytotoxicity of BM CAR αβ T cells armed with two cytokine receptors of a truncated NKG2D chimera by FACS detection. Figure 18 shows the calculated T cell proliferation from the same experiment. The data show that truncated NKG2D chimeric cytokine receptor arming improved the cytotoxicity and proliferation of BM CAR T cells.

[0329] Figure 19 shows the long-term cytotoxicity of BM CAR γδ T cells armed with anti-MIC-A / B scFv chimeric dual cytokine receptors by FACS detection. Figure 20 shows calculated T cell proliferation from the same experiment. The data show that anti-MIC-A / B scFv chimeric cytokine receptor arming improved the cytotoxicity and proliferation of BM CAR T cells.

[0330] Figure 22 shows the long-term cytotoxicity of Construct 185 CAR αβ T cells armed with two cytokine receptors of the NKG2D chimera (anti-GPC3 CAR αβ T cells) by FACS detection. Figure 23 shows the calculated T cell proliferation from the same experiment. The data show that arming two cytokine receptors of the NKG2D chimera improved the cytotoxicity and proliferation of Construct 185 CAR CAR T cells.

[0331] Figures 24 and 26 show the long-term cytotoxicity of CTL-019 CAR (anti-CD19 CAR) γδ T cells or αβ T cells armed with two cytokine receptors of the NKG2D chimera, as detected by FACS. Figures 25 and 27 show calculated T cell proliferation from the same experiments. The data show that NKG2D chimeric cytokine receptor arming improved the cytotoxicity and proliferation of CTL-019 CAR T cells.

[0332] Figure 28 shows the long-term cytotoxicity of AS67190 CAR αβ T cells (anti-CD33 CAR αβ T cells) armed with two cytokine receptors of the NKG2D chimera by FACS detection. Figure 29 shows the calculated T cell proliferation from the same experiment. The data show that arming two cytokine receptors of the NKG2D chimera improved the cytotoxicity and proliferation of AS67190 CAR T cells.

[0333] Figure 30 shows the long-term cytotoxicity of BM CAR γδ T cells armed with two cytokine receptors of the TIGIT chimera by FACS detection. Figure 31 shows the calculated T cell proliferation from the same experiment. The data show that TIGIT chimera cytokine receptor arming improved the cytotoxicity and proliferation of BM CAR T cells.

[0334] Figure 33 shows the long-term cytotoxicity of AS67190 CAR γδ T cells armed with two cytokine receptors of a SIRP-α chimera as detected by FACS. Figure 34 shows calculated T cell proliferation from the same experiment. The data show that SIRP-α chimera cytokine receptor arming improved the cytotoxicity and proliferation of AS67190 CAR T cells.

[0335] Figure 37 shows the long-term cytotoxicity of BM CAR αβ T cells armed with NKG2D-deficient or constitutively active chimeric dual cytokine receptors by FACS detection. Figure 38 shows calculated T cell proliferation from the same experiment. The data show that NKG2D is important for chimeric cytokine receptor arming.

[0336] 6.8. Example 8 - In Vivo Safety and Efficacy Evaluation The in vivo anti-tumor activity of exemplary anti-GPC3 CAR-T cells (γδ T cells or αβ T cells) was evaluated in a huh7 xenograft model. Briefly, on day 0, 3 million (3 × 10 6 ) huh7 cells were subcutaneously implanted into NOD / SCID IL-2RγC null (NSG) mice. Ten days after tumor inoculation, mice were treated as follows: 1 × 10 6 Armed CAR-γδ T or mock T cells or phosphate-buffered saline (PBS) were injected intravenously. Tumor size was measured twice weekly with a caliper, and tumor volume was calculated using the formula V = 1 / 2 (length × width). The mean tumor burden in control mice was 2,000 mm. 3 Once the mice reached 100% T cell proliferation, they were euthanized and T cell proliferation was monitored by FACS analysis of plasma collected from the blood.

[0337] For toxicity assessment, animals were monitored daily for clinical signs and body weight, and blood (0.2 mL) was collected weekly to detect the mouse humanized cytokine profile (GM-CSF, IFN-γ, and TNF-α).

[0338] Figure 21 shows the anti-tumor effects of anti-GPC3 CAR γδ T cells or anti-GPC3 CAR γδ T cells armed with two NKG2D chimeric cytokine receptors in a huh7 xenograft model. Unarmed CAR-γδ T cells suppressed tumor growth, along with CAR-γδ T cells armed with chimeric cytokine receptors. Notably, mice treated with unarmed CAR-γδ T cells achieved tumor-free status but slowly rebounded, whereas mice treated with CAR-γδ T cells armed with two NKG2D chimeric cytokine receptors achieved tumor-free status and remained healthy and tumor-free throughout the experimental observation period.

[0339] Figure 32 shows the anti-tumor effects of anti-GPC3 CAR γδ T cells or anti-GPC3 CAR γδ T cells armed with a TIGIT chimeric cytokine receptor in a huh7 xenograft model. Unarmed CAR-γδ T cells suppressed tumor growth, along with CAR-γδ T cells armed with a chimeric cytokine receptor. Notably, mice treated with unarmed CAR-γδ T cells were slightly suppressed, while mice treated with CAR-γδ T cells armed with two TIGIT chimeric cytokine receptors achieved tumor-free status.

[0340] In summary, CAR-γδ T cells armed with chimeric cytokine receptors have proven to be effective and safe in tumor therapy, as shown by in vitro efficacy, in vivo efficacy, and safety studies.

[0341] From the foregoing, it will be understood that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope provided herein. All references mentioned above are incorporated herein by reference in their entirety.

[0342] Sequence Listing SEQ ID NO: 1 (CD8α signal peptide amino acid sequence) MALPVTALLLPLALLLHAARP SEQ ID NO:2 (GM-CSF signal peptide amino acid sequence) MWLQSLLLLGTVACSIS SEQ ID NO:3 (IL-15 signal peptide amino acid sequence) MRISKPHLRSISIQCYLCLLLNSHFLTEA SEQ ID NO:4 (FLAG tag amino acid sequence) DYKDDDDK SEQ ID NO: 5 (HA tag amino acid sequence) YPYDVPDYA SEQ ID NO:6 (NKG2D 81-216 amino acid sequence) SLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO:7 (TpoR 478-582 amino acid sequence) SDPTRVETATETAWISLVTALHLVLGLSAVLGLLLLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPL SEQ ID NO:8 (IL7Rα 316-459 amino acid sequence) ARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ SEQ ID NO:9 (IL9R-1 310-464 amino acid sequence) MFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSS SEQ ID NO: 10 (IL9R-2 391-464 amino acid sequence) DVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSS SEQ ID NO: 11 (IL9R-3 310-337, 391-464 amino acid sequence) MFFQPLYSVHNGNFQTWMGAHGAGVLLSDVLP...

Claims

1. (i) A chimeric cytokine receptor, comprising: (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof; (b) a first transmembrane domain, and (c) Cytokine receptor intracellular domain The chimeric cytokine receptor comprising: (ii) optionally, a functional exogenous receptor, (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) Intracellular signaling domain The functional exogenous receptor comprising: expressing, immune effector cells.

2. The immune effector cell of claim 1 , wherein the first extracellular antigen-binding domain binds to an antigen expressed on the surface of a tumor cell.

3. (i) the first extracellular antigen-binding domain is derived from NKG2D or a truncated NKG2D, or a variant thereof, optionally wherein the first extracellular antigen-binding domain is derived from the extracellular domain (ECD) of NKG2D or a truncated NKG2D, and optionally wherein the first extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6, 156 or 157, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6, 156 or 157; (ii) the first extracellular antigen domain comprises an antibody or antigen-binding fragment thereof that binds to an NKG2D ligand or a variant thereof, optionally wherein the NKG2D ligand is selected from the group consisting of MICA / B, ULBP-1, ULBP-2, 5, 6 and ULBP-3, and optionally wherein the antibody or antigen-binding fragment thereof comprises one or more scFvs or one or more sdAbs that bind to the NKG2D ligand; (iii) the first extracellular antigen domain is derived from TIGIT or a variant thereof, optionally the first extracellular antigen domain is derived from the ECD of TIGIT, and optionally the first extracellular antigen binding domain comprises an amino acid sequence of SEQ ID NO: 145 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 145; or (iv) the first extracellular antigen domain is derived from SIRP-α or a variant thereof, optionally wherein the first extracellular antigen domain is derived from the ECD of SIRP-α, and optionally wherein the first extracellular antigen binding domain comprises an amino acid sequence of SEQ ID NO: 147, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 147; The immune effector cell of claim 1.

4. The immune effector cell of claim 1 , wherein the first transmembrane domain comprises a Janus kinase (JAK) binding domain.

5. The immune effector cell of claim 4, wherein the JAK binding domain is derived from the group consisting of EPOR, GHR, TPOR or a variant thereof.

6. 6. The immune effector cell of claim 5, wherein the JAK-binding domain is derived from TPOR and comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

7.

7. 2. The immune effector cell of claim 1, wherein the cytokine receptor intracellular domain is derived from one or more cytokine receptors selected from the group consisting of IL2Rα, IL7Rα, IL9R-1, IL9R-2, IL9R-3, IL12Rβ1, IL12Rβ2, IL15Rα, IL15Rβ-1, IL15Rβ-2, IL15Rβ-3, IL18Rα, IL18Rβ, IL21R-1, IL21R-2, IL21R-3, IL10R2, IL22R1, IL23R-1, IL23R-2, IL23R-3, IL27Rα, gp130, IL31RA, OSMRβ, IL36R, IL1RAcP, GM-CSFRα, GM-CSFRβ-1, GM-CSFRβ-2, or variants and combinations thereof.

8. Thanks for watching and LOVELYα 20β1、1120β2、115Xα、1150β1、1 150β22、150β33、150000α、18Xβ、 LO2001、02002、102003 、LOVESY2、LOVESYS3、DYSHAKα、DYS DAβ11、DHASHSA β22、DYBDA1、NDASHY2、 LOVE LOVE α0012 β1 LOVE LOVEα 150α、LOVEXα0150β11、LOVESα01 CHAβ22、ROSHYXα002101、ROSHYα1 202、ROSHYXα002003、0000000 32、ROSHYXαSYSα、ROYSYαSHASHα DASHβ11、CH7DASHDASHβ02、S12B 1015α、1120β10150β110120 β10150β2、1020β100010、1 12Sβ100102、1002β100213 、X12β100232、1020β15S HANAα、C12B150000000000000000000000000000000000000000000 HISTORY β22、112β20155α、112 β20150β11、0120β20150β02、1 LO2BYβ200011、LOY20000000 2、012β2000203、12000 32、C12β2DXα、112β25 STICSβ11、C12β2DASSASIβ22、1 150α00101、1500000.000 LOVEFAX1003、LOVE000202、 C1FISHαSCIENCEα、C15αSCIENCEβ 11、CH15αFASJSβ22、#150β1 LOVE10、150β01002020、1150β 10000003、10000000000000000000000000 150β15DXα、1500000 ROβ11、1500000000000000000000000000000000000000000000 β####101、#########2、115Bβ0200203、4 50β200302、1150 β#2DXα、C15Bβ22DXSβ01、C15β02 CHRISTIAN β22、1021 LOYYYOY2、LOYYOY15 SHYα、ROY21JOY1 SHβ11、SY211S DAβ22、LOY212023 ROY2、LOY2020 α、ROYOK20SHASHβ. 1、ROY202052 、LOYYYYYYYYYYYYYY2、1 LO2100000000000000000000000000000000000000000000 yet? 1SHASHYB11、142 1SHASHIS β22、123 FAMOUS SATISα、ROYISH 2DHASHIS β11 THIS 2DHASHIS β22、CHASYα. LOVE LOVE2、LO120β10LOVE 22、012β200002、1 RO1FISHαLOVE LOVE22、150β. 1000020、1050β020 LOVE20、LOVE210LOVE 22、LOYYYYYYYYYYYYYYYYYYY2、 LOY20000000000000000000000,000,000,000,000,000,0 THIS IS YOUR LIFE αLOVE LOVE2、FIRST β1 LOVE YOU 、DYSIS β221 LOVE LOVE、 LOVE LOVEα0020β 2. There is nothing wrong with this Thanks for watching the seven-year-old.

9. 8. The immune effector of claim 7, wherein the cytokine receptor intracellular domain comprises one or more amino acid sequences selected from SEQ ID NOs: 8-28, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NOs: 8-28.

10. 2. The immune effector cell of claim 1, wherein the functional exogenous receptor is a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), or a T cell antigen coupler (TAC)-like chimeric receptor.

11. The immune effector cell of claim 10, wherein the CAR or TCR binds to a tumor-associated antigen, and optionally the tumor-associated antigen is selected from the group consisting of GPC3, AFP, claudin 18.2, CD19, CD20, CD22, BCMA, GD2, DLL3, MSLN, CD30, CLL1, and CD33.

12. 2. The immune effector cell of claim 1, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, and optionally, the intracellular signaling domain comprises a costimulatory signaling domain.

13. 11. The immune effector cell of claim 10, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 29, 135, 141, 143 or 149, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 29, 135, 141, 143 or 149.

14. 2. The immune effector cell of claim 1, comprising an amino acid sequence of SEQ ID NO: 30-134, 136-140, 142, 144, 146 or 148, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30-134, 136-140, 142, 144, 146 or 148.

15. The immune effector cell of claim 1, wherein the immune effector cell is a T cell, a natural killer (NK) cell, an NK T cell, a macrophage, a peripheral blood mononuclear cell (PBMC), a monocyte, a neutrophil or an eosinophil, and optionally, the T cell is a cytotoxic T cell, a helper T cell, a natural killer T cell, an αβ T cell or a γδ T cell.

16. (i) a first region encoding a chimeric cytokine receptor, the chimeric cytokine receptor comprising: (a) a first extracellular antigen-binding domain derived from NKG2D, a truncated NKG2D, an antibody or antigen-binding fragment thereof targeting an NKG2D ligand, TIGIT or SIRP-α, or a variant thereof; (b) a first transmembrane domain, and (c) Cytokine receptor intracellular domain The first region includes: (ii) optionally, a second region encoding a functional exogenous receptor, said functional exogenous receptor comprising: (a) a second extracellular antigen-binding domain, (b) a second transmembrane domain, and (c) Intracellular signaling domain the second region including An isolated nucleic acid comprising:

17. A vector comprising the nucleic acid of claim 16.

18. A pharmaceutical composition comprising an immune effector cell of claim 1, a nucleic acid of claim 16, or a vector of claim 17, and a pharma- ceutical acceptable carrier.

19. 20. A medicament for treating a disease or disorder in a subject, comprising an effective amount of the pharmaceutical composition of claim 18.

20. 20. The method of claim 19, wherein the disease or disorder is cancer, an inflammatory disease or an autoimmune disease, optionally wherein the cancer is a solid cancer or a blood cancer, optionally wherein the cancer is liver cancer, lymphoma, acute myeloid leukemia (AML) or chronic myelogenous leukemia (CML).

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